Spatially distinguished, multiplex nucleic acid analysis of biological specimens

ABSTRACT

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 371 of International Application No.PCT/EP2016/057355, filed on Apr. 6, 2016, which claims the benefit ofU.S. Provisional Application No. 62/145,874.

BACKGROUND

One of every four men will die of cancer. Further statistics from theAmerican Cancer Society predict that one of every five women will sufferthe same fate. Treatments are available for many cancers. However,success for most relies on early detection.

Cancer is now said to be a disease of the genome. Many oncologists andcancer researchers hope that advances in genomic analysis tools willprovide early detection and a path to treatment. However, these toolsare more prominent in research labs having not yet matured to the levelof being readily available to the vast majority of oncologists.Improvements are needed.

It has been said that at the time of diagnosis, all cancer patients aremosaics. They are mosaics because they have at least two distinctgenomes: the genome they were born with, and the genome that theyunwillingly acquired via cancer. Furthermore, as tumors grow, distinctpopulations of cancer cells become apparent. Leading to even morecomplex mosaics within the tumor. This cancer cell heterogeneity oftenresults in subpopulations of cells that respond differently to cancertherapies. The end result is often an initial positive response of onesubpopulation of cells, resulting in the observation of the patient'stumor shrinking, only to be followed by regrowth of tumor tissue, and insome cases metastasis. Despite early detection of the tumor, aninability to identify the subpopulation of cells that are resistant tothe treatment can result in loss of time needed to treat an aggressivecancer. This creates adverse consequences for the patient bothemotionally and physically.

There is a need for genomic tools that can distinguish subpopulations ofcancer cells in tumors. The present disclosure addresses this need andprovides other advantages as well.

BRIEF SUMMARY

The present disclosure provides a method for spatially tagging nucleicacids of a biological specimen. The method can include steps of (a)providing a solid support comprising a plurality of different nucleicacid probes that are randomly located on the solid support, wherein thedifferent nucleic acid probes each includes a barcode sequence that isdifferent from the barcode sequence of other randomly located probes onthe solid support; (b) performing a nucleic acid detection reaction onthe solid support to locate the barcode sequences on the solid support;(c) contacting a biological specimen with the solid support that has therandomly located probes; (d) hybridizing the randomly located probes totarget nucleic acids from portions of the biological specimen that areproximal to the randomly located probes; and (e) modifying the randomlylocated probes that are hybridized to the target nucleic acids, therebyproducing modified probes that include the barcode sequences and atarget specific modification, thereby spatially tagging the nucleicacids of the biological specimen.

This disclosure further provides a method for spatially tagging nucleicacids of a biological specimen, the method including steps of (a)attaching different nucleic acid probes to a solid support to producerandomly located probes on the solid support, wherein the differentnucleic acid probes each includes a barcode sequence, and wherein eachof the randomly located probes includes different barcode sequences fromother randomly located probes on the solid support; (b) performing anucleic acid detection reaction on the solid support to determine thebarcode sequences of the randomly located probes on the solid support;(c) contacting a biological specimen with the solid support that has therandomly located probes; (d) hybridizing the randomly located probes totarget nucleic acids from portions of the biological specimen that areproximal to the randomly located probes; and (e) extending the randomlylocated probes to produce extended probes that include the barcodesequences and sequences from the target nucleic acids, thereby spatiallytagging the nucleic acids of the biological specimen.

Also provided is a method for spatially tagging nucleic acids of abiological specimen that includes the steps of (a) providing a pluralityof nucleic acid primers attached to a solid support, wherein the nucleicacid primers in the plurality include a universal primer sequence thatis common to the nucleic acid primers in the plurality; (b) binding apopulation of nucleic acid probes to the plurality of nucleic acidprimers, wherein the nucleic acid probes include a universal primerbinding sequence that hybridizes to the universal primer sequence, atarget capture sequence and a barcode sequence that differs from barcodesequences of other nucleic acid probes in the population, therebyattaching the different nucleic acid probes at randomly locatedpositions on the solid support; (c) amplifying the different nucleicacid probes by extension of the nucleic acid primers, thereby producingnucleic acid clusters having copies of the barcode sequence and targetcapture sequence at the randomly located positions on the solid support;(d) performing a sequencing reaction to determine the barcode sequencesat the randomly located positions on the solid support; (e) contacting abiological specimen with the nucleic acid clusters on the solid support;(f) hybridizing the target capture sequences of the clusters to targetnucleic acids from portions of the biological specimen that are proximalto the clusters; and (g) extending the target capture sequences toproduce extended probes that include sequences from the target nucleicacids and the copies of the barcode sequences, thereby tagging thenucleic acids of the biological specimen.

This disclosure further provides a method for spatially tagging nucleicacids of a biological specimen, the method including steps of (a)providing an array of beads on a solid support, wherein differentnucleic acid probes are attached to different beads in the array,wherein the different nucleic acid probes each include a barcodesequence, wherein each bead includes a different barcode sequence fromother beads on the solid support, and wherein each of the differentnucleic acid probes includes a target capture sequence; (b) performing adecoder probe hybridization reaction on the solid support to determinethe barcode sequences at the randomly located probes on the solidsupport; (c) contacting a biological specimen with the array of beads;(d) hybridizing the different nucleic acid probes to target nucleicacids from portions of the biological specimen that are proximal to thebeads; and (e) extending the different nucleic acid probes to produceextended probes that include sequences from the target nucleic acids andthe barcode sequences, thereby tagging the nucleic acids of thebiological specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagrammatic representation of steps and reagents thatcan be used to generate barcoded oligo dT probes on an Illumina flowcell, create extended barcoded probes having mRNA sequences andreleasing the extended probes from the flow cell.

FIG. 2 shows data indicating the availability of oligo dT capturesequences on probes after bridge amplification of the probes andrestriction enzyme digest with BspH1 to remove one of the primer bindingsites used for bridge amplification.

FIG. 3 shows sequencing metrics of the flow cell described in Example 1and shown in FIG. 2.

FIG. 4 the number of unique barcodes determined in 21 tiles of the flowcell described in Example 1 and shown in FIG. 2.

FIG. 5 shows an image of cells captured on a patterned flow cell (PanelA) and cell count data (Panel B).

FIG. 6 shows cells that remain adhered to a flow cell in differentconditions.

FIG. 7 shows a diagrammatic representation of steps and reagents used tocreate probes attached to a gel (Panel A), a diagrammatic representationof steps and reagents used to capture target nucleic acids using thegel-attached probes and fluorescently label the probes (Panel B), and animage created by the fluorescently labeled target nucleic acidsfollowing capture by the probes and removal of the tissue from the gel.

FIG. 8 shows a diagrammatic representation of steps and reagents used tocapture target nucleic acids using BeadArray™-attached probes andfluorescently label the probes (Panel A), and an image created by thefluorescently labeled target nucleic acids following capture by theprobes and removal of the tissue from the BeadArray™.

DETAILED DESCRIPTION

The present disclosure provides compositions, apparatus and methods forpreserving spatial information when performing multiplex nucleic acidanalyses of biological specimens. A variety of tools are available formultiplex nucleic acid analyses including, for example, nucleic acidmicroarrays and so-called “next generation” sequencing platforms. Suchtools allow for parallel detection of very large and complex collectionsof nucleic acids, including for example, DNA collections that representall or nearly all of the genetic material of an organism (i.e. the‘genome’), RNA (or cDNA) collections that represent all or nearly all ofthe complement of expressed genes (i.e. the ‘transcriptome’) for anorganism, and in some cases the collections can include several genomesand/or transcriptomes from several different organisms (e.g. ametabolome or biome from a community or ecosystem). Although these toolsprovide a vast amount of information about what nucleic acid sequencesare present in a biological specimen being evaluated, they do notinherently distinguish where any particular nucleic acid resided in thebiological specimen. Indeed the vast majority of samples applied tomultiplex nucleic acid analysis tools are homogenates derived frommixtures of many different cells from a biological specimen. As aresult, spatial information is lost and the results obtained from thesetools constitute an average transcriptome or average genome for thespecimen, important differences between individual cells being lost.

In particular embodiments, the present disclosure provides new anduseful modifications to existing multiplex nucleic acid analysis toolsto allow for the preservation of spatial information for biologicalspecimens from which the nucleic acids are obtained. For example, solidsupports that are usually used for multiplex sequencing-by-synthesis(SBS) techniques can be modified for use in capturing and spatiallytagging nucleic acids from a biological specimen. In an alternativeexample, arrays of beads, such as those used for genotyping or geneexpression analysis, can be used for capturing and spatially taggingnucleic acids from a biological specimen. As set forth in examplesbelow, the solid supports used for an SBS or BeadArray™ platformcommercialized by Illumina (San Diego, Calif.) can be modified forspatial tagging. However, it will be understood that any of a variety ofsolid supports can be made and used in accordance with the teachingherein. The spatially tagged nucleic acids can be removed from the solidsupport, pooled together and attached to a second solid support fordetection in any of a variety of multiplex nucleic acid analysis systemsincluding, for example, a sequencing platform or microarray platform setforth herein.

The spatial information provided by a method, composition or apparatusherein can include, for example, the location of one or more cells in atissue (or other specimen) that has a particular allele at one or morelocus (e.g. a genotype), has a particular structural variation in thegenome (e.g. fusion, insertion, deletion, rearrangement etc.), has aparticular epigenetic signature (e.g. methylation), expresses aparticular gene, expresses a particular allele of a gene, expresses aparticular splice variant of a gene or the like. In addition toidentifying nucleic acids according to their spatial location in abiological specimen, a method, composition or apparatus of the presentdisclosure can be used to quantify one or more nucleic acids accordingto spatial location. For example, the spatial information for one ormore cells in a tissue (or other specimen) can include the amount of aparticular allele or chromosomal region in a genome (e.g. ploidy); theamount of epigenetic modification of a genetic locus (e.g. methylation);expression level for a particular gene, allele or splice variant; or thelike. The amounts can be absolute amounts or relative amounts inaccordance with similar measurements obtained in the art for mixed ornon-spatially tagged samples.

A method set forth herein can be used for localized detection of anucleic acid in a biological specimen. In some embodiments, a method canbe used for identifying or characterizing all of the transcriptome orgenome of a biological specimen. Alternatively, a method can be used toidentify or characterize only a part of a specimen's transcriptome orgenome. A subset of transcripts or genes evaluated in a method hereincan be related to a particular disease or condition.

A method set forth herein can be used for localized or spatial detectionof nucleic acids, whether DNA or RNA, in a biological specimen. Thus oneor more RNA or DNA molecules can be located with respect to its nativeposition or location within a cell or tissue or other biologicalspecimen. For example, one or more nucleic acids can be localized to acell or group of adjacent cells, or type of cell, or to particularregions of areas within a tissue sample. The native location or positionof individual RNA or DNA molecules can be determined using a method,apparatus or composition of the present disclosure.

Terms used herein will be understood to take on their ordinary meaningin the relevant art unless specified otherwise. Several terms usedherein and their meanings are set forth below.

As used herein, the term “amplicon,” when used in reference to a nucleicacid, means the product of copying the nucleic acid, wherein the producthas a nucleotide sequence that is the same as or complementary to atleast a portion of the nucleotide sequence of the nucleic acid. Anamplicon can be produced by any of a variety of amplification methodsthat use the nucleic acid, or an amplicon thereof, as a templateincluding, for example, polymerase extension, polymerase chain reaction(PCR), rolling circle amplification (RCalif.), multiple displacementamplification (MDA), ligation extension, or ligation chain reaction. Anamplicon can be a nucleic acid molecule having a single copy of aparticular nucleotide sequence (e.g. a PCR product) or multiple copiesof the nucleotide sequence (e.g. a concatameric product of RCalif.). Afirst amplicon of a target nucleic acid is typically a complementarycopy. Subsequent amplicons are copies that are created, after generationof the first amplicon, from the target nucleic acid or from the firstamplicon. A subsequent amplicon can have a sequence that issubstantially complementary to the target nucleic acid or substantiallyidentical to the target nucleic acid.

As used herein, the term “array” refers to a population of features orsites that can be differentiated from each other according to relativelocation. Different molecules that are at different sites of an arraycan be differentiated from each other according to the locations of thesites in the array. An individual site of an array can include one ormore molecules of a particular type. For example, a site can include asingle target nucleic acid molecule having a particular sequence or asite can include several nucleic acid molecules having the same sequence(and/or complementary sequence, thereof). The sites of an array can bedifferent features located on the same substrate. Exemplary featuresinclude without limitation, wells in a substrate, beads (or otherparticles) in or on a substrate, projections from a substrate, ridges ona substrate or channels in a substrate. The sites of an array can beseparate substrates each bearing a different molecule. Differentmolecules attached to separate substrates can be identified according tothe locations of the substrates on a surface to which the substrates areassociated or according to the locations of the substrates in a liquidor gel. Exemplary arrays in which separate substrates are located on asurface include, without limitation, those having beads in wells.

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other. Forexample, an analyte, such as a nucleic acid, can be attached to amaterial, such as a gel or solid support, by a covalent or non-covalentbond. A covalent bond is characterized by the sharing of pairs ofelectrons between atoms. A non-covalent bond is a chemical bond thatdoes not involve the sharing of pairs of electrons and can include, forexample, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilicinteractions and hydrophobic interactions.

As used herein, the term “barcode sequence” is intended to mean a seriesof nucleotides in a nucleic acid that can be used to identify thenucleic acid, a characteristic of the nucleic acid, or a manipulationthat has been carried out on the nucleic acid. The barcode sequence canbe a naturally occurring sequence or a sequence that does not occurnaturally in the organism from which the barcoded nucleic acid wasobtained. A barcode sequence can be unique to a single nucleic acidspecies in a population or a barcode sequence can be shared by severaldifferent nucleic acid species in a population. For example, eachnucleic acid probe in a population can include different barcodesequences from all other nucleic acid probes in the population.Alternatively, each nucleic acid probe in a population can includedifferent barcode sequences from some or most other nucleic acid probesin a population. For example, each probe in a population can have abarcode that is present for several different probes in the populationeven though the probes with the common barcode differ from each other atother sequence regions along their length. In particular embodiments,one or more barcode sequences that are used with a biological specimenare not present in the genome, transcriptome or other nucleic acids ofthe biological specimen. For example, barcode sequences can have lessthan 80%, 70%, 60%, 50% or 40% sequence identity to the nucleic acidsequences in a particular biological specimen.

As used herein, the term “biological specimen” is intended to mean oneor more cell, tissue, organism or portion thereof. A biological specimencan be obtained from any of a variety of organisms. Exemplary organismsinclude, but are not limited to, a mammal such as a rodent, mouse, rat,rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog,primate (i.e. human or non-human primate); a plant such as Arabidopsisthaliana, corn, sorghum, oat, wheat, rice, canola, or soybean; an algaesuch as Chlamydomonas reinhardtii; a nematode such as Caenorhabditiselegans; an insect such as Drosophila melanogaster, mosquito, fruit fly,honey bee or spider; a fish such as zebrafish; a reptile; an amphibiansuch as a frog or Xenopus laevis; a Dictyostelium discoideum; a fungisuch as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a Plasmodium falciparum.Target nucleic acids can also be derived from a prokaryote such as abacterium, Escherichia coli, Staphylococci or Mycoplasma pneumoniae; anarchae; a virus such as Hepatitis C virus or human immunodeficiencyvirus; or a viroid. Specimens can be derived from a homogeneous cultureor population of the above organisms or alternatively from a collectionof several different organisms, for example, in a community orecosystem.

As used herein, the term “cleavage site” is intended to mean a locationin a nucleic acid molecule that is susceptible to bond breakage. Thelocation can be specific to a particular chemical, enzymatic or physicalprocess that results in bond breakage. For example, the location can bea nucleotide that is abasic or a nucleotide that has a base that issusceptible to being removed to create an abasic site. Examples ofnucleotides that are susceptible to being removed include uracil and8-oxo-guanine as set forth in further detail herein below. The locationcan also be at or near a recognition sequence for a restrictionendonuclease such as a nicking enzyme.

As used herein, the term “cluster,” when used in reference to nucleicacids, refers to a population of the nucleic acids that is attached to asolid support to form a feature or site. The nucleic acids are generallymembers of a single species, thereby forming a monoclonal cluster. A“monoclonal population” of nucleic acids is a population that ishomogeneous with respect to a particular nucleotide sequence. Clustersneed not be monoclonal. Rather, for some applications, a cluster can bepredominantly populated with amplicons from a first nucleic acid and canalso have a low level of contaminating amplicons from a second nucleicacid. For example, when an array of clusters is to be used in adetection application, an acceptable level of contamination would be alevel that does not impact signal to noise or resolution of thedetection technique in an unacceptable way. Accordingly, apparentclonality will generally be relevant to a particular use or applicationof an array made by the methods set forth herein. Exemplary levels ofcontamination that can be acceptable at an individual cluster include,but are not limited to, at most 0.1%, 0.5%, 1%, 5%, 10%, 5 25%, or 35%contaminating amplicons. The nucleic acids in a cluster are generallycovalently attached to a solid support, for example, via their 5′ ends,but in some cases other attachment means are possible. The nucleic acidsin a cluster can be single stranded or double stranded. In some but notall embodiments, clusters are made by a solid-phase amplification methodknown as bridge amplification. Exemplary configurations for clusters andmethods for their production are set forth, for example, in U.S. Pat.No. 5,641,658; U.S. Patent Publ. No. 2002/0055100; U.S. Pat. No.7,115,400; U.S. Patent Publ. No. 2004/0096853; U.S. Patent Publ. No.2004/0002090; U.S. Patent Publ. No. 2007/0128624; and U.S. Patent Publ.No. 2008/0009420, each of which is incorporated herein by reference.

As used herein, the term “different”, when used in reference to nucleicacids, means that the nucleic acids have nucleotide sequences that arenot the same as each other. Two or more nucleic acids can havenucleotide sequences that are different along their entire length.Alternatively, two or more nucleic acids can have nucleotide sequencesthat are different along a substantial portion of their length. Forexample, two or more nucleic acids can have target nucleotide sequenceportions that are different for the two or more molecules while alsohaving a universal sequence portion that is the same on the two or moremolecules. Two beads can be different from each other by virtue of beingattached to different nucleic acids.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “extend,” when used in reference to a nucleicacid, is intended to mean addition of at least one nucleotide oroligonucleotide to the nucleic acid. In particular embodiments one ormore nucleotides can be added to the 3′ end of a nucleic acid, forexample, via polymerase catalysis (e.g. DNA polymerase, RNA polymeraseor reverse transcriptase). Chemical or enzymatic methods can be used toadd one or more nucleotide to the 3′ or 5′ end of a nucleic acid. One ormore oligonucleotides can be added to the 3′ or 5′ end of a nucleicacid, for example, via chemical or enzymatic (e.g. ligase catalysis)methods. A nucleic acid can be extended in a template directed manner,whereby the product of extension is complementary to a template nucleicacid that is hybridized to the nucleic acid that is extended.

As used herein, the term “feature” means a location in an array for aparticular species of molecule. A feature can contain only a singlemolecule or it can contain a population of several molecules of the samespecies. Features of an array are typically discrete. The discretefeatures can be contiguous or they can have spaces between each other.The size of the features and/or spacing between the features can varysuch that arrays can be high density, medium density or lower density.High density arrays are characterized as having sites separated by lessthan about 15 μm. Medium density arrays have sites separated by about 15to 30 μm, while low density arrays have sites separated by greater than30 μm. An array useful herein can have, for example, sites that areseparated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. Anapparatus or method of the present disclosure can be used to detect anarray at a resolution sufficient to distinguish sites at the abovedensities or density ranges.

As used herein, the term “fluidic mixture” is intended to mean two ormore different items that are simultaneously present in a solution.Typically, the two or more items are freely diffusible in the solution.The two or more items can be different types of items (e.g. a nucleicacid and a protein which are different types of molecules) or they canbe different species of the same type of items (e.g. two nucleic acidmolecules having different sequences). Exemplary items that can be in afluidic mixture include, but are not limited to, molecules, cells orbeads.

As used herein, the term “flow cell” is intended to mean a vessel havinga chamber where a reaction can be carried out, an inlet for deliveringreagents to the chamber and an outlet for removing reagents from thechamber. In some embodiments the chamber is configured for detection ofthe reaction that occurs in the chamber. For example, the chamber caninclude one or more transparent surfaces allowing optical detection ofbiological specimens, optically labeled molecules, or the like in thechamber. Exemplary flow cells include, but are not limited to those usedin a nucleic acid sequencing apparatus such as flow cells for the GenomeAnalyzer®, MiSeq®, NextSeq® or HiSeq® platforms commercialized byIllumina, Inc. (San Diego, Calif.); or for the SOLiD™ or Ion Torrent™sequencing platform commercialized by Life Technologies (Carlsbad,Calif.). Exemplary flow cells and methods for their manufacture and useare also described, for example, in WO 2014/142841 A1; U.S. Pat. App.Pub. No. 2010/0111768 A1 and U.S. Pat. No. 8,951,781, each of which isincorporated herein by reference.

As used herein, the term “gel” is intended to mean a semi-rigid materialthat is permeable to liquids and gases. Typically, gel material canswell when liquid is taken up and can contract when liquid is removed bydrying. Exemplary gels include, but are not limited to those having acolloidal structure, such as agarose; polymer mesh structure, such asgelatin; or cross-linked polymer structure, such as polyacrylamide, SFA(see, for example, US Pat. App. Pub. No. 2011/0059865 A1, which isincorporated herein by reference) or PAZAM (see, for example, US Pat.App. Publ. No. 2014/0079923 A1, which is incorporated herein byreference). Particularly useful gel material will conform to the shapeof a well or other concave feature where it resides.

As used herein, the terms “nucleic acid” and “nucleotide” are intendedto be consistent with their use in the art and to include naturallyoccurring species or functional analogs thereof. Particularly usefulfunctional analogs of nucleic acids are capable of hybridizing to anucleic acid in a sequence specific fashion or capable of being used asa template for replication of a particular nucleotide sequence.Naturally occurring nucleic acids generally have a backbone containingphosphodiester bonds. An analog structure can have an alternate backbonelinkage including any of a variety of those known in the art. Naturallyoccurring nucleic acids generally have a deoxyribose sugar (e.g. foundin deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found inribonucleic acid (RNA)). A nucleic acid can contain nucleotides havingany of a variety of analogs of these sugar moieties that are known inthe art. A nucleic acid can include native or non-native nucleotides. Inthis regard, a native deoxyribonucleic acid can have one or more basesselected from the group consisting of adenine, thymine, cytosine orguanine and a ribonucleic acid can have one or more bases selected fromthe group consisting of uracil, adenine, cytosine or guanine. Usefulnon-native bases that can be included in a nucleic acid or nucleotideare known in the art. The terms “probe” or “target,” when used inreference to a nucleic acid or sequence of a nucleic acid, are intendedas semantic identifiers for the nucleic acid or sequence in the contextof a method or composition set forth herein and does not necessarilylimit the structure or function of the nucleic acid or sequence beyondwhat is otherwise explicitly indicated. The terms “probe” and “target”can be similarly applied to other analytes such as proteins, smallmolecules, cells or the like.

As used herein, the term “pitch,” when used in reference to features ofan array, is intended to refer to the center-to-center spacing foradjacent features. A pattern of features can be characterized in termsof average pitch. The pattern can be ordered such that the coefficientof variation around the average pitch is small or the pattern can berandom in which case the coefficient of variation can be relativelylarge. In either case, the average pitch can be, for example, at leastabout 10 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 100 μm or more.Alternatively or additionally, the average pitch can be, for example, atmost about 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm 0.1 μm or less. Of course,the average pitch for a particular pattern of features can be betweenone of the lower values and one of the upper values selected from theranges above.

As used herein, the term “poly T or poly A,” when used in reference to anucleic acid sequence, is intended to mean a series of two or morethiamine (T) or adenine (A) bases, respectively. A poly T or poly A caninclude at least about 2, 5, 8, 10, 12, 15, 18, 20 or more of the T or Abases, respectively. Alternatively or additionally, a poly T or poly Acan include at most about, 30, 20, 18, 15, 12, 10, 8, 5 or 2 of the T orA bases, respectively.

As used herein, the term “random” can be used to refer to the spatialarrangement or composition of locations on a surface. For example, thereare at least two types of order for an array described herein, the firstrelating to the spacing and relative location of features (also called“sites”) and the second relating to identity or predetermined knowledgeof the particular species of molecule that is present at a particularfeature. Accordingly, features of an array can be randomly spaced suchthat nearest neighbor features have variable spacing between each other.Alternatively, the spacing between features can be ordered, for example,forming a regular pattern such as a rectilinear grid or hexagonal grid.In another respect, features of an array can be random with respect tothe identity or predetermined knowledge of the species of analyte (e.g.nucleic acid of a particular sequence) that occupies each featureindependent of whether spacing produces a random pattern or orderedpattern. An array set forth herein can be ordered in one respect andrandom in another. For example, in some embodiments set forth herein asurface is contacted with a population of nucleic acids under conditionswhere the nucleic acids attach at sites that are ordered with respect totheir relative locations but ‘randomly located’ with respect toknowledge of the sequence for the nucleic acid species present at anyparticular site. Reference to “randomly distributing” nucleic acids atlocations on a surface is intended to refer to the absence of knowledgeor absence of predetermination regarding which nucleic acid will becaptured at which location (regardless of whether the locations arearranged in an ordered pattern or not).

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g. due to porosity) but will typically be sufficiently rigid that thesubstrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.Particularly useful solid supports for some embodiments are locatedwithin a flow cell apparatus. Exemplary flow cells are set forth infurther detail herein.

As used herein, the term “spatial tag” is intended to mean a nucleicacid having a sequence that is indicative of a location. Typically, thenucleic acid is a synthetic molecule having a sequence that is not foundin one or more biological specimen that will be used with the nucleicacid. However, in some embodiments the nucleic acid molecule can benaturally derived or the sequence of the nucleic acid can be naturallyoccurring, for example, in a biological specimen that is used with thenucleic acid. The location indicated by a spatial tag can be a locationin or on a biological specimen, in or on a solid support or acombination thereof. A barcode sequence can function as a spatial tag.

As used herein, the term “tissue” is intended to mean an aggregation ofcells, and, optionally, intercellular matter. Typically the cells in atissue are not free floating in solution and instead are attached toeach other to form a multicellular structure. Exemplary tissue typesinclude muscle, nerve, epidermal and connective tissues.

As used herein, the term “universal sequence” refers to a series ofnucleotides that is common to two or more nucleic acid molecules even ifthe molecules also have regions of sequence that differ from each other.A universal sequence that is present in different members of acollection of molecules can allow capture of multiple different nucleicacids using a population of universal capture nucleic acids that arecomplementary to the universal sequence. Similarly, a universal sequencepresent in different members of a collection of molecules can allow thereplication or amplification of multiple different nucleic acids using apopulation of universal primers that are complementary to the universalsequence. Thus, a universal capture nucleic acid or a universal primerincludes a sequence that can hybridize specifically to a universalsequence. Target nucleic acid molecules may be modified to attachuniversal adapters, for example, at one or both ends of the differenttarget sequences.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides a method for spatially tagging nucleicacids of a biological specimen. The method can include the steps of (a)attaching different nucleic acid probes to a solid support to producerandomly located probes on the solid support, wherein the differentnucleic acid probes each includes a barcode sequence, and wherein eachof the randomly located probes includes different barcode sequences fromother randomly located probes on the solid support; (b) performing anucleic acid detection reaction on the solid support to determine thebarcode sequences of the randomly located probes on the solid support;(c) contacting a biological specimen with the solid support that has therandomly located probes; (d) hybridizing the randomly located probes totarget nucleic acids from portions of the biological specimen that areproximal to the randomly located probes; and (e) extending the randomlylocated probes to produce extended probes that include the barcodesequences and sequences from the target nucleic acids, thereby spatiallytagging the nucleic acids of the biological specimen.

Any of a variety of solid supports can be used in a method, compositionor apparatus of the present disclosure. Particularly useful solidsupports are those used for nucleic acid arrays. Examples include glass,modified glass, functionalized glass, inorganic glasses, microspheres(e.g. inert and/or magnetic particles), plastics, polysaccharides,nylon, nitrocellulose, ceramics, resins, silica, silica-based materials,carbon, metals, an optical fiber or optical fiber bundles, polymers andmultiwell (e.g. microtiter) plates. Exemplary plastics include acrylics,polystyrene, copolymers of styrene and other materials, polypropylene,polyethylene, polybutylene, polyurethanes and Teflon™. Exemplarysilica-based materials include silicon and various forms of modifiedsilicon.

In particular embodiments, a solid support can be within or part of avessel such as a well, tube, channel, cuvette, Petri plate, bottle orthe like. A particularly useful vessel is a flow-cell, for example, asdescribed in WO 2014/142841 A1; U.S. Pat. App. Pub. No. 2010/0111768 A1and U.S. Pat. No. 8,951,781 or Bentley et al., Nature 456:53-59 (2008),each of which is incorporated herein by reference. Exemplary flow-cellsare those that are commercially available from Illumina, Inc. (SanDiego, Calif.) for use with a sequencing platform such as a GenomeAnalyzer®, MiSeq®, NextSeq® or HiSeq® platform. Another particularlyuseful vessel is a well in a multiwell plate or microtiter plate.

Optionally, a solid support can include a gel coating. Attachment ofnucleic acids to a solid support via a gel is exemplified by flow cellsavailable commercially from Illumina Inc. (San Diego, Calif.) ordescribed in US Pat. App. Pub. Nos. 2011/0059865 A1, 2014/0079923 A1, or2015/0005447 A1; or PCT Publ. No. WO 2008/093098, each of which isincorporated herein by reference. Exemplary gels that can be used in themethods and apparatus set forth herein include, but are not limited to,those having a colloidal structure, such as agarose; polymer meshstructure, such as gelatin; or cross-linked polymer structure, such aspolyacrylamide, SFA (see, for example, US Pat. App. Pub. No.2011/0059865 A1, which is incorporated herein by reference) or PAZAM(see, for example, US Pat. App. Publ. Nos. 2014/0079923 A1, or2015/0005447 A1, each of which is incorporated herein by reference).

In some embodiments, a solid support can be configured as an array offeatures to which nucleic acids can be attached. The features can bepresent in any of a variety of desired formats. For example, thefeatures can be wells, pits, channels, ridges, raised regions, pegs,posts or the like. In some embodiments, the features can contain beads.However, in particular embodiments the features need not contain a beador particle. Exemplary features include wells that are present insubstrates used for commercial sequencing platforms sold by 454LifeSciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent(a subsidiary of Life Technologies, Carlsbad Calif.). Other substrateshaving wells include, for example, etched fiber optics and othersubstrates described in U.S. Pat Nos. 6,266,459; 6,355,431; 6,770,441;6,859,570; 6,210,891; 6,258,568; 6,274,320; US Pat app. Publ. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; 2010/0282617 A1 orPCT Publication No. WO 00/63437, each of which is incorporated herein byreference. In some embodiments, wells of a substrate can include gelmaterial (with or without beads) as set forth in US Pat. App. Publ. No.2014/0243224 A1, which is incorporated herein by reference.

The features on a solid support can be metal features on a non-metallicsurface such as glass, plastic or other materials exemplified above. Ametal layer can be deposited on a surface using methods known in the artsuch as wet plasma etching, dry plasma etching, atomic layer deposition,ion beam etching, chemical vapor deposition, vacuum sputtering or thelike. Any of a variety of commercial instruments can be used asappropriate including, for example, the FlexAl®, OpAL®, lonfab 300plus®,or Optofab 30000® systems (Oxford Instruments, UK). A metal layer canalso be deposited by e-beam evaporation or sputtering as set forth inThornton, Ann. Rev. Mater. Sci. 7:239-60 (1977), which is incorporatedherein by reference. Metal layer deposition techniques, such as thoseexemplified above, can be combined with photolithography techniques tocreate metal regions or patches on a surface. Exemplary methods forcombining metal layer deposition techniques and photolithographytechniques are provided in U.S. Pat. No. 8,895,249 or US Pat App. Pub.No. 2014/0243224 A1, each of which is incorporated herein by reference.

Features can appear on a solid support as a grid of spots or patches.The features can be located in a repeating pattern or in an irregular,non-repeating pattern. Particularly useful repeating patterns arehexagonal patterns, rectilinear patterns, grid patterns, patterns havingreflective symmetry, patterns having rotational symmetry, or the like.Asymmetric patterns can also be useful. The pitch can be the samebetween different pairs of nearest neighbor features or the pitch canvary between different pairs of nearest neighbor features.

High density arrays are characterized as having average pitch of lessthan about 15 μm. Medium density arrays have average pitch of about 15to 30 μm, while low density arrays have average pitch greater than 30μm. An array useful in the invention can have average pitch that is lessthan 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm. The average pitchvalues and ranges set forth above or elsewhere herein are intended to beapplicable to ordered arrays or random arrays.

In particular embodiments, features on a solid support can each have anarea that is larger than about 100 nm², 250 nm², 500 nm², 1 μm², 2.5μm², 5 μm², 10 μm², 100 μm², or 500 μm². Alternatively or additionally,features can each have an area that is smaller than about 1 mm², 500μm², 100 μm², 25 μm2, 10 μm², 5 μm², 1 μm², 500 nm², or 100 nm². Theabove ranges can describe the apparent area of a bead or other particleon a solid support when viewed or imaged from above.

In particular embodiments, a solid support can include a collection ofbeads or other particles. The particles can be suspended in a solutionor they can be located on the surface of a substrate. Examples of arrayshaving beads located on a surface include those wherein beads arelocated in wells such as a BeadChip array (Illumina Inc., San DiegoCalif.), substrates used in sequencing platforms from 454 LifeSciences(a subsidiary of Roche, Basel Switzerland) or substrates used insequencing platforms from Ion Torrent (a subsidiary of LifeTechnologies, Carlsbad Calif.). Other solid supports having beadslocated on a surface are described in U.S. Pat. Nos. 6,266,459;6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; or 6,274,320; USPat. App. Publ. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1;or 2010/0282617 A1 or PCT Publication No. WO 00/63437, each of which isincorporated herein by reference. Several of the above referencesdescribe methods for attaching nucleic acid probes to beads prior toloading the beads in or on a solid support. As such, the collection ofbeads can include different beads each having a unique probe attached.It will however, be understood that the beads can be made to includeuniversal primers, and the beads can then be loaded onto an array,thereby forming universal arrays for use in a method set forth herein.As set forth previously herein, the solid supports typically used forbead arrays can be used without beads. For example, nucleic acids, suchas probes or primers can be attached directly to the wells or to gelmaterial in wells. Thus, the above references are illustrative ofmaterials, compositions or apparatus that can be modified for use in themethods and compositions set forth herein.

Accordingly, a solid support used in a method set forth herein caninclude an array of beads, wherein different nucleic acid probes areattached to different beads in the array. In this embodiment, each beadcan be attached to a different nucleic acid probe and the beads can berandomly distributed on the solid support in order to effectively attachthe different nucleic acid probes to the solid support. Optionally, thesolid support can include wells having dimensions that accommodate nomore than a single bead. In such a configuration, the beads may beattached to the wells due to forces resulting from the fit of the beadsin the wells. It is also possible to use attachment chemistries oradhesives to hold the beads in the wells.

Nucleic acid probes that are attached to beads can include barcodesequences. A population of the beads can be configured such that eachbead is attached to only one type of barcode and many different beadseach with a different barcode are present in the population. In thisembodiment, randomly distributing the beads to a solid support willresult in randomly locating the nucleic acid probes (and theirrespective barcode sequences) on the solid support. In some cases therecan be multiple beads with the same barcode sequence such that there isredundancy in the population. Randomly distributing a redundantpopulation of beads on a solid support that has a capacity that isgreater than the number of unique barcodes in the bead population willresult in redundancy of barcodes on the solid support. Alternatively,the number of different barcodes in a population of beads can exceed thecapacity of the solid support in order to produce an array that is notredundant with respect to the population of barcodes on the solidsupport. The capacity of the solid support will be determined in someembodiments by the number of features (e.g. single-bead occupancy wells)that attach or otherwise accommodate a bead.

A solid support can include, or can be made by the methods set forthherein to attach, a plurality of different nucleic acid probes. Forexample, a solid support can include at least 10, 100, 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁶, 1×10⁹ or more different probes.Alternatively or additionally, a solid support can include at most1×10⁹, 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 100, or fewer differentprobes. It will be understood that each of the different probes can bepresent in several copies, for example, when the probes have beenamplified to form a cluster. Thus, the above ranges can describe thenumber of different nucleic acid clusters on a solid support. It willalso be understood that the above ranges can describe the number ofdifferent barcodes, target capture sequences, or other sequence elementsset forth herein as being unique to particular nucleic acid probes.Alternatively or additionally, the ranges can describe the number ofextended probes or modified probes created on a solid support using amethod set forth herein.

Features, may be present on a solid support prior to contacting thesolid support with nucleic acid probes. For example, in embodimentswhere probes are attached to a support via hybridization to primers, theprimers can be attached at the features, whereas interstitial areasoutside of the features substantially lack any of the primers. Nucleicacid probes can be captured at preformed features on a solid support,and optionally amplified on the solid support, using methods set forthin U.S. Pat. Nos. 8,895,249, 8,778,849, or US Pat App. Pub. No.2014/0243224 A1, each of which is incorporated herein by reference.Alternatively, a solid support may have a lawn of primers or mayotherwise lack features. In this case, a feature can be formed by virtueof attachment of a nucleic acid probe on the solid support. Optionally,the captured nucleic acid probe can be amplified on the solid supportsuch that the resulting cluster becomes a feature. Although attachmentis exemplified above as capture between a primer and a complementaryportion of a probe, it will be understood that capture moieties otherthan primers can be present at pre-formed features or as a lawn. Otherexemplary capture moieties include, but are not limited to, chemicalmoieties capable of reacting with a nucleic acid probe to create acovalent bond or receptors capable of biding non-covalently to a ligandon a nucleic acid probe.

A step of attaching nucleic acid probes to a solid support can becarried out by providing a fluid that contains a mixture of differentnucleic acid probes and contacting this fluidic mixture with the solidsupport. The contact can result in the fluidic mixture being in contactwith a surface to which many different nucleic acid probes from thefluidic mixture will attach. Thus, the probes have random access to thesurface (whether the surface has pre-formed features configured toattach the probes or a uniform surface configured for attachment).Accordingly, the probes can be randomly located on the solid support.

The total number and variety of different probes that end up attached toa surface can be selected for a particular application or use. Forexample, in embodiments where a fluidic mixture of different nucleicacid probes is contacted with a solid support for purposes of attachingthe probes to the support, the number of different probe species canexceed the occupancy of the solid support for probes. Thus, the numberand variety of different probes that attach to the solid support can beequivalent to the probe occupancy of the solid support. Alternatively,the number and variety of different probe species on the solid supportcan be less than the occupancy (i.e. there will be redundancy of probespecies such that the solid support may contain multiple features havingthe same probe species). Such redundancy can be achieved, for example,by contacting the solid support with a fluidic mixture that contains anumber and variety of probe species that is substantially lower than theprobe occupancy of the solid support.

Attachment of the nucleic acid probes can be mediated by hybridizationof the nucleic acid probes to complementary primers that are attached tothe solid support, chemical bond formation between a reactive moiety onthe nucleic acid probe and the solid support (examples are set forth inU.S. Pat. Nos. 8,895,249, 8,778,849, or US Pat App. Pub. No.2014/0243224 A1, each of which is incorporated herein by reference),affinity interactions of a moiety on the nucleic acid probe with a solidsupport-bound moiety (e.g. between known receptor-ligand pairs such asstreptavidinbiotin, antibody-epitope, lectin-carbohydrate and the like),physical interactions of the nucleic acid probes with the solid support(e.g. hydrogen bonding, ionic forces, van der Waals forces and thelike), or other interactions known in the art to attach nucleic acids tosurfaces.

In some embodiments, attachment of a nucleic acid probe is non-specificwith regard to any sequence differences between the nucleic acid probeand other nucleic acid probes that are or will be attached to the solidsupport. For example, different probes can have a universal sequencethat complements surface-attached primers or the different probes canhave a common moiety that mediates attachment to the surface.Alternatively, each of the different probes (or a subpopulation ofdifferent probes) can have a unique sequence that complements a uniqueprimer on the solid support or they can have a unique moiety thatinteracts with one or more different reactive moiety on the solidsupport. In such cases, the unique primers or unique moieties can,optionally, be attached at predefined locations in order to selectivelycapture particular probes, or particular types of probes, at therespective predefined locations.

One or more features on a solid support can each include a singlemolecule of a particular probe. The features can be configured, in someembodiments, to accommodate no more than a single nucleic acid probemolecule. However, whether or not the feature can accommodate more thanone nucleic acid probe molecule, the feature may nonetheless include nomore than a single nucleic acid probe molecule. Alternatively, anindividual feature can include a plurality of nucleic acid probemolecules, for example, an ensemble of nucleic acid probe moleculeshaving the same sequence as each other. In particular embodiments, theensemble can be produced by amplification from a single nucleic acidprobe template to produce amplicons, for example, as a cluster attachedto the surface.

A method set forth herein can use any of a variety of amplificationtechniques. Exemplary techniques that can be used include, but are notlimited to, polymerase chain reaction (PCR), rolling circleamplification (RCalif.), multiple displacement amplification (MDA), orrandom prime amplification (RPA). In some embodiments the amplificationcan be carried out in solution, for example, when features of an arrayare capable of containing amplicons in a volume having a desiredcapacity. Preferably, an amplification technique used in a method of thepresent disclosure will be carried out on solid phase. For example, oneor more primer species (e.g. universal primers for one or more universalprimer binding site present in a nucleic acid probe) can be attached toa solid support. In PCR embodiments, one or both of the primers used foramplification can be attached to a solid support (e.g. via a gel).Formats that utilize two species of primers attached to a solid supportare often referred to as bridge amplification because double strandedamplicons form a bridge-like structure between the two surface attachedprimers that flank the template sequence that has been copied. Exemplaryreagents and conditions that can be used for bridge amplification aredescribed, for example, in U.S. Pat. Nos. 5,641,658, 7,115,400, or8,895,249; or U.S. Pat. Publ. Nos. 2002/0055100 A1, 2004/0096853 A1,2004/0002090 A1, 2007/0128624 A1 or 2008/0009420 A1, each of which isincorporated herein by reference. Solid-phase PCR amplification can alsobe carried out with one of the amplification primers attached to a solidsupport and the second primer in solution. An exemplary format that usesa combination of a surface attached primer and soluble primer is theformat used in emulsion PCR as described, for example, in Dressman etal., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05/010145, orU.S. Pat. App. Publ. Nos. 2005/0130173 A1 or 2005/0064460 A1, each ofwhich is incorporated herein by reference. Emulsion PCR is illustrativeof the format and it will be understood that for purposes of the methodsset forth herein the use of an emulsion is optional and indeed forseveral embodiments an emulsion is not used.

RCA techniques can be modified for use in a method of the presentdisclosure. Exemplary components that can be used in an RCA reaction andprinciples by which RCA produces amplicons are described, for example,in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US Pat. App. Publ.No. 2007/0099208 A1, each of which is incorporated herein by reference.Primers used for RCA can be in solution or attached to a solid support.The primers can be one or more of the universal primers describedherein.

MDA techniques can be modified for use in a method of the presentdisclosure. Some basic principles and useful conditions for MDA aredescribed, for example, in Dean et al., Proc Natl. Acad. Sci. USA99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003);Walker et al., Molecular Methods for Virus Detection, Academic Press,Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); U.S. Pat.Nos. 5,455,166; 5,130,238; and 6,214,587, each of which is incorporatedherein by reference. Primers used for MDA can be in solution or attachedto a solid support at an amplification site. Again, the primers can beone or more of the universal primers described herein.

In particular embodiments a combination of the above-exemplifiedamplification techniques can be used. For example, RCA and MDA can beused in a combination wherein RCA is used to generate a concatamericamplicon in solution (e.g. using solution-phase primers). The ampliconcan then be used as a template for MDA using primers that are attachedto a solid support (e.g. universal primers). In this example, ampliconsproduced after the combined RCA and MDA steps will be attached to thesolid support.

Nucleic acid probes that are used in a method set forth herein orpresent in an apparatus or composition of the present disclosure caninclude barcode sequences, and for embodiments that include a pluralityof different nucleic acid probes, each of the probes can include adifferent barcode sequence from other probes in the plurality. Barcodesequences can be any of a variety of lengths. Longer sequences cangenerally accommodate a larger number and variety of barcodes for apopulation. Generally, all probes in a plurality will have the samelength barcode (albeit with different sequences), but it is alsopossible to use different length barcodes for different probes. Abarcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20 or morenucleotides in length. Alternatively or additionally, the length of thebarcode sequence can be at most 20, 15, 12, 10, 8, 6, 4 or fewernucleotides. Examples of barcode sequences that can be used are setforth, for example in, US Pat. App. Publ. No. 2014/0342921 A1 and U.S.Pat. No. 8,460,865, each of which is incorporated herein by reference.

A method of the present disclosure can include a step of performing anucleic acid detection reaction on a solid support to determine barcodesequences of nucleic acid probes that are located on the solid support.In many embodiments the probes are randomly located on the solid supportand the nucleic acid detection reaction provides information to locateeach of the different probes. Exemplary nucleic acid detection methodsinclude, but are not limited to nucleic acid sequencing of a probe,hybridization of nucleic acids to a probe, ligation of nucleic acidsthat are hybridized to a probe, extension of nucleic acids that arehybridized to a probe, extension of a first nucleic acid that ishybridized to a probe followed by ligation of the extended nucleic acidto a second nucleic acid that is hybridized to the probe, or othermethods known in the art such as those set forth in U.S. Pat. Nos.8,288,103 or 8,486,625, each of which is incorporated herein byreference.

Sequencing techniques, such as sequencing-by-synthesis (SBS) techniques,are a particularly useful method for determining barcode sequences. SBScan be carried out as follows. To initiate a first SBS cycle, one ormore labeled nucleotides, DNA polymerase, SBS primers etc., can becontacted with one or more features on a solid support (e.g. feature(s)where nucleic acid probes are attached to the solid support). Thosefeatures where SBS primer extension causes a labeled nucleotide to beincorporated can be detected. Optionally, the nucleotides can include areversible termination moiety that terminates further primer extensiononce a nucleotide has been added to the SBS primer. For example, anucleotide analog having a reversible terminator moiety can be added toa primer such that subsequent extension cannot occur until a deblockingagent is delivered to remove the moiety. Thus, for embodiments that usereversible termination, a deblocking reagent can be delivered to thesolid support (before or after detection occurs). Washes can be carriedout between the various delivery steps. The cycle can then be repeated ntimes to extend the primer by n nucleotides, thereby detecting asequence of length n. Exemplary SBS procedures, fluidic systems anddetection platforms that can be readily adapted for use with acomposition, apparatus or method of the present disclosure aredescribed, for example, in Bentley et al., Nature 456:53-59 (2008), PCTPubl. Nos. WO 91/06678, WO 04/018497 or WO 07/123744; U.S. Pat. Nos.7,057,026, 7,329,492, 7,211,414, 7,315,019 or 7,405,281, and US Pat.App. Publ. No. 2008/0108082, each of which is incorporated herein byreference.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); or U.S. Pat. Nos. 6,210,891,6,258,568 or 6,274,320, each of which is incorporated herein byreference). In pyrosequencing, released PPi can be detected by beingimmediately converted to adenosine triphosphate (ATP) by ATPsulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons. Thus, the sequencing reaction can bemonitored via a luminescence detection system. Excitation radiationsources used for fluorescence based detection systems are not necessaryfor pyrosequencing procedures. Useful fluidic systems, detectors andprocedures that can be used for application of pyrosequencing toapparatus, compositions or methods of the present disclosure aredescribed, for example, in PCT Pat. App. Publ. No. WO2012/058096, USPat. App. Publ. No. 2005/0191698 A1, or U.S. Pat. Nos. 7,595,883 or7,244,559, each of which is incorporated herein by reference.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); or U.S.Pat. Nos. 5,599,675 or 5,750,341, each of which is incorporated hereinby reference. Some embodiments can include sequencing-by-hybridizationprocedures as described, for example, in Bains et al., Journal ofTheoretical Biology 135(3), 303-7 (1988); Drmanac et al., NatureBiotechnology 16, 54-58 (1998); Fodor et al., Science 251 (4995),767-773 (1995); or PCT Pat. App. Publ. No. WO 1989/10977, each of whichis incorporated herein by reference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, target nucleic acids (oramplicons thereof) that are present at sites of an array are subjectedto repeated cycles of oligonucleotide delivery and detection.Compositions, apparatus or methods set forth herein or in referencescited herein can be readily adapted for sequencing-by-ligation orsequencing-by-hybridization procedures. Typically, the oligonucleotidesare fluorescently labeled and can be detected using fluorescencedetectors similar to those described with regard to SBS proceduresherein or in references cited herein.

Some sequencing embodiments can utilize methods involving the real-timemonitoring of DNA polymerase activity. For example, nucleotideincorporations can be detected through fluorescence resonance energytransfer (FRET) interactions between a fluorophore-bearing polymeraseand y-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs).Techniques and reagents for FRET-based sequencing are described, forexample, in Levene et al. Science 299, 682-686 (2003); Lundquist et al.Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci.USA 105, 1176-1181 (2008), each of which is incorporated herein byreference.

Some sequencing embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, CT, a Life Technologies and Thermo Fishersubsidiary) or sequencing methods and systems described in US Pat app.Publ. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or US2010/0282617 A1, each of which is incorporated herein by reference.

Nucleic acid hybridization techniques are also useful method fordetermining barcode sequences. In some cases combinatorial hybridizationmethods can be used such as those used for decoding of multiplex beadarrays (see e.g. U.S. Pat. No. 8,460,865, which is incorporated hereinby reference). Such methods utilize labelled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence. Ahybridization reaction can be carried out using decoder probes havingknown labels such that the location where the labels end up on the solidsupport identifies the nucleic acid probes according to rules of nucleicacid complementarity. In some cases, pools of many different probes withdistinguishable labels are used, thereby allowing a multiplex decodingoperation. The number of different barcodes determined in a decodingoperation can exceed the number of labels used for the decodingoperation. For example, decoding can be carried out in several stageswhere each stage constitutes hybridization with a different pool ofdecoder probes. The same decoder probes can be present in differentpools but the label that is present on each decoder probe can differfrom pool to pool (i.e. each decoder probe is in a different “state”when in different pools). Various combinations of these states andstages can be used to expand the number of barcodes that can be decodedwell beyond the number of distinct labels available for decoding. Suchcombinatorial methods are set forth in further detail in U.S. Pat. No.8,460,865 or Gunderson et al., Genome Research 14:870-877 (2004), eachof which is incorporated herein by reference.

A method of the present disclosure can include a step of contacting abiological specimen with a solid support that has nucleic acid probesattached thereto. In some embodiments the nucleic acid probes arerandomly located on the solid support. The identity and location of thenucleic acid probes may have been decoded prior to contacting thebiological specimen with the solid support. Alternatively, the identityand location of the nucleic acid probes can be determined aftercontacting the solid support with the biological specimen.

In some embodiments the biological specimen is one or more cells. Thecell(s) can be individual and free from any tissue or multicellularstructure at the time contact is made with the solid support. Forexample, the cell(s) can be present in a fluid (e.g. when a plurality ofdifferent cells are present the fluid can be a fluidic mixture of thedifferent cells) and the fluid can be contacted with the solid supportto which the different probes are attached. Any of a variety of cellscan be used including, for example, those from a prokaryote, archae oreukaryote. One or more cells used in a method, composition or apparatusof the present disclosure can be a single celled organisms or from amulticellular organism. Exemplary organisms from which one or more cellcan be obtained include, but are not limited to a mammal, plant, algae,nematode, insect, fish, reptile, amphibian, fungi or Plasmodiumfalciparum. Exemplary species are set forth previously herein or knownin the art.

Embodiments of the present disclosure can also use one or moresubcellular components as a biological specimen. For example a fluidicmixture can include one or more nuclei, golgi apparatus, mitochondria,chloroplasts, membrane fractions, vesicles, endoplasmic reticulum, orother components known in the art. Other useful types of biologicalspecimens are one or more viruses or a viroids.

It will be understood that a biological specimen can be a homogeneousculture or population of the above cells, subcellular components,viruses or viroids. Alternatively the biological specimen can be anon-homogenous collection of cells, subcellular components, viruses orviroids, for example, derived from several different organisms in acommunity or ecosystem. An exemplary community is the collection ofbacteria present in the digestive system, lung or other organ of amulticellular organism such as a mammal.

One or more cells, subcellular components, viruses or viroids that arecontacted with a solid support in a method set forth herein can beattached to the solid support. Attachment can be achieved using methodsknown in the art such as those exemplified herein with respect toattachment of nucleic acids to a solid support. In some embodiments,attachment is selective for specific types of cells, subcellularcomponents, viruses or viroids. For example, the solid support caninclude antibodies or other receptors that are selective for epitopes orligands present on one or a subset of different cells, subcellularcomponents, viruses or viroids present in a fluidic mixture. In otherembodiments, the attachment of cells, subcellular components, viruses orviroids can be mediated by non-selective moieties such as chemicalmoieties that are broadly reactive.

In particular embodiments, one or more cells, subcellular components,viruses or viroids that have been contacted with a solid support can belysed to release target nucleic acids. Lysis can be carried out usingmethods known in the art such as those that employ one or more ofchemical treatment, enzymatic treatment, electroporation, heat,hypotonic treatment, sonication or the like. Exemplary lysis techniquesare set forth in Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Ed., Cold Spring Harbor Laboratory, New York (2001) and inAnsubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1999).

In some embodiments the biological specimen is a tissue section. Thetissue can be derived from a multicellular organism such as thoseexemplified above in regard to cells. A tissue section can be contactedwith a solid support, for example, by laying the tissue on the surfaceof the solid support. The tissue can be freshly excised from an organismor it may have been previously preserved for example by freezing,embedding in a material such as paraffin (e.g. formalin fixed paraffinembedded samples), formalin fixation, infiltration, dehydration or thelike. Optionally, a tissue section can be attached to a solid support,for example, using techniques and compositions exemplified herein withregard to attaching nucleic acids, cells, viruses, beads or the like toa solid support. As a further option, a tissue can be permeabilized andthe cells of the tissue lysed when the tissue is in contact with a solidsupport. Any of a variety of treatments can be used such as those setforth above in regard to lysing cells. Target nucleic acids that arereleased from a tissue that is permeabilized can be captured by nucleicacid probes on the surface.

A tissue can be prepared in any convenient or desired way for its use ina method, composition or apparatus herein. Fresh, frozen, fixed orunfixed tissues can be used. A tissue can be fixed or embedded usingmethods described herein or known in the art.

A tissue sample for use herein, can be fixed by deep freezing attemperature suitable to maintain or preserve the integrity of the tissuestructure, e.g. less than −20° C. In another example, a tissue can beprepared using formalin-fixation and paraffin embedding (FFPE) methodswhich are known in the art. Other fixatives and/or embedding materialscan be used as desired. A fixed or embedded tissue sample can besectioned, i.e. thinly sliced, using known methods. For example, atissue sample can be sectioned using a chilled microtome or cryostat,set at a temperature suitable to maintain both the structural integrityof the tissue sample and the chemical properties of the nucleic acids inthe sample.

In some embodiments, a tissue sample will be treated to remove embeddingmaterial (e.g. to remove paraffin or formalin) from the sample prior torelease, capture or modification of nucleic acids. This can be achievedby contacting the sample with an appropriate solvent (e.g. xylene andethanol washes). Treatment can occur prior to contacting the tissuesample with a solid support set forth herein or the treatment can occurwhile the tissue sample is on the solid support. Exemplary methods formanipulating tissues for use with solid supports to which nucleic acidsare attached are set forth in US Pat. App. Publ. No. 2014/0066318 A1,which is incorporated herein by reference.

The thickness of a tissue sample or other biological specimen that iscontacted with a solid support in a method, composition or apparatus setforth herein can be any suitable thickness desired. In representativeembodiments, the thickness will be at least 0.1 μm, 0.25 μm, 0.5 μm,0.75 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm or thicker. Alternatively oradditionally, the thickness of a biological specimen that is contactedwith a solid support will be no more than 100 μm, 50 μm, 10 μm, 5 μm, 1μm, 0.5 μm, 0.25 μm, 0.1 μm or thinner.

A particularly relevant source for a biological specimen is a humanbeing. The specimen can be derived from an organ, including for example,an organ of the musculoskeletal system such as muscle, bone, tendon orligament; an organ of the digestive system such as salivary gland,pharynx, esophagus, stomach, small intestine, large intestine, liver,gallbladder or pancreas; an organ of the respiratory system such aslarynx, trachea, bronchi, lungs or diaphragm; an organ of the urinarysystem such as kidney, ureter, bladder or urethra; a reproductive organsuch as ovary, fallopian tube, uterus, vagina, placenta, testicle,epididymis, vas deferens, seminal vesicle, prostate, penis or scrotum;an organ of the endocrine system such as pituitary gland, pineal gland,thyroid gland, parathyroid gland, or adrenal gland; an organ of thecirculatory system such as heart, artery, vein or capillary; an organ ofthe lymphatic system such as lymphatic vessel, lymph node, bone marrow,thymus or spleen; an organ of the central nervous system such as brain,brainstem, cerebellum, spinal cord, cranial nerve, or spinal nerve; asensory organ such as eye, ear, nose, or tongue; or an organ of theintegument such as skin, subcutaneous tissue or mammary gland. In someembodiments, a biological specimen is obtained from a bodily fluid orexcreta such as blood, lymph, tears, sweat, saliva, semen, vaginalsecretion, ear wax, fecal matter or urine.

A specimen from a human can be considered (or suspected) healthy ordiseased when used. In some cases, two specimens can be used: a firstbeing considered diseased and a second being considered as healthy (e.g.for use as a healthy control). Any of a variety of conditions can beevaluated, including but not limited to, an autoimmune disease, cancer,cystic fibrosis, aneuploidy, pathogenic infection, psychologicalcondition, hepatitis, diabetes, sexually transmitted disease, heartdisease, stroke, cardiovascular disease, multiple sclerosis or musculardystrophy. Particularly relevant conditions are genetic conditions orconditions associated with pathogens having identifiable geneticsignatures.

As set forth above, a flow cell provides a convenient apparatus for usein a method set forth herein. For example, a flow cell is a convenientapparatus for housing a solid support that will be treated with multiplefluidic reagents such as the repeated fluidic deliveries used for somenucleic acid sequencing protocols or some nucleic acid hybridizationprotocols. In some embodiments, a biological specimen can be deliveredto a solid support in a flow cell, for example, when a fluidic mixtureof cells, subcellular components, viruses or viroids is delivered to thesolid support. In some embodiments it may be preferable to open a flowcell to expose a solid support inside or to remove the solid supportfrom the flow cell in order to allow convenient delivery of a biologicalspecimen to the solid support. For example, opening the flow cell orremoving the solid support can allow a user or robotic device to lay atissue section on the solid support. The opening of a flow cell orremoval of a solid support from a flow cell can be temporary. Thus, theflow cell can subsequently be closed or the solid support returned tothe flow cell to proceed with one or more subsequent steps of a methodset forth herein.

In some embodiments, a flow cell can have a construction that allows itto be opened or taken apart. For example the flow cell can be in aclosed state while performing a sequencing reaction, for example todecode barcodes. Then the flow cell can be taken apart so that tissuecan be placed on the flow cell surface. The flow cell can be heldtogether by adhesive such that one or more surface can be removed toopen it. For example, a flow cell can have a spacer with adhesivesurfaces on the top or bottom (akin to single-sided or double-sidedsticky tape) and this spacer can occur between two solid supports. Oneor both of the solid supports can be configured to attach nucleic acidsand support a biological specimen as set forth herein. The spacer canhave open regions (e.g. created by laser cutting of the spacer material)that create fluidic channels bound by the two solid supports and thespacer. Thus, one or both of the solid supports can be non-permanentlyadhered to the spacer to allow one or both of them to be removed toallow access to the surface when placing a tissue or other specimenthereon.

A nucleic acid probe used in a composition, apparatus or method setforth herein can include a target capture moiety. In particularembodiments, the target capture moiety is a target capture sequence. Thetarget capture sequence is generally complementary to a target sequencesuch that target capture occurs by formation of a probe-target hybridcomplex. A target capture sequence can be any of a variety of lengthsincluding, for example, lengths exemplified above in the context ofbarcode sequences.

In multiplex embodiments, a plurality of different nucleic acid probescan include different target capture sequences that hybridize todifferent target nucleic acid sequences from a biological specimen.Different target capture sequences can be used to selectively bind toone or more desired target nucleic acids from a biological specimen. Insome cases, the different nucleic acid probes can include a targetcapture sequence that is common to all or a subset of the probes on asolid support. For example, the nucleic acid probes on a solid supportcan have a poly A or poly T sequence. Such probes or amplicons thereofcan hybridize to mRNA molecules, cDNA molecules or amplicons thereofthat have poly A or poly T tails. Although the mRNA or cDNA species willhave different target sequences, capture will be mediated by the commonpoly A or poly T sequence regions.

Any of a variety of target nucleic acids can be captured and analyzed ina method set forth herein including, but not limited to, messenger RNA(mRNA), copy DNA (cDNA), genomic DNA (gDNA), ribosomal RNA (rRNA) ortransfer RNA (tRNA). Particular target sequences can be selected fromdatabases and appropriate capture sequences designed using techniquesand databases known in the art.

Other target capture moieties that are useful include, for example, themoieties set forth herein as useful for attaching nucleic acid probes toa solid support.

A method set forth herein can include a step of hybridizing nucleic acidprobes, that are on a solid support, to target nucleic acids that arefrom portions of the biological specimen that are proximal to theprobes. Generally, a target nucleic acid will diffuse from a region ofthe biological specimen to an area of the solid support that is inproximity with that region of the specimen. Here the target nucleic acidwill interact with nucleic acid probes that are proximal to the regionof the specimen from which the target nucleic acid was released. Atarget-probe hybrid complex can form where the target nucleic acidencounters a complementary target capture sequence on a nucleic acidprobe. The location of the target-probe hybrid complex will generallycorrelate with the region of the biological specimen from where thetarget nucleic acid was derived. In multiplex embodiments, the solidsupport will include a plurality of nucleic acid probes, the biologicalspecimen will release a plurality of target nucleic acids and aplurality of target-probe hybrids will be formed on the solid support.The sequences of the target nucleic acids and their locations on thesupport will provide spatial information about the nucleic acid contentof the biological specimen. Although the example above is described inthe context of target nucleic acids that are released from a biologicalspecimen, it will be understood that the target nucleic acids need notbe released. Rather, the target nucleic acids may remain in contact withthe biological specimen, for example, when they are attached to anexposed surface of the biological specimen in a way that the targetnucleic acids can also bind to appropriate nucleic acid probes on thesolid support.

A method of the present disclosure can include a step of extending solidsupport-attached probes to which target nucleic acids are hybridized. Inembodiments where the probes include barcode sequences, the resultingextended probes will include the barcode sequences and sequences fromthe target nucleic acids (albeit in complementary form). The extendedprobes are thus spatially tagged versions of the target nucleic acidsfrom the biological specimen. The sequences of the extended probesidentify what nucleic acids are in the biological specimen and where inthe biological specimen the target nucleic acids are located. It will beunderstood that other sequence elements that are present in the nucleicacid probes can also be included in the extended probes. Such elementsinclude, for example, primer binding sites, cleavage sites, other tagsequences (e.g. sample identification tags), capture sequences,recognition sites for nucleic acid binding proteins or nucleic acidenzymes, or the like.

Extension of probes can be carried out using methods exemplified hereinor otherwise known in the art for amplification of nucleic acids orsequencing of nucleic acids. In particular embodiments one or morenucleotides can be added to the 3′ end of a nucleic acid, for example,via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reversetranscriptase). Chemical or enzymatic methods can be used to add one ormore nucleotide to the 3′ or 5′ end of a nucleic acid. One or moreoligonucleotides can be added to the 3′ or 5′ end of a nucleic acid, forexample, via chemical or enzymatic (e.g. ligase catalysis) methods. Anucleic acid can be extended in a template directed manner, whereby theproduct of extension is complementary to a template nucleic acid that ishybridized to the nucleic acid that is extended. In some embodiments, aDNA primer is extended by a reverse transcriptase using an RNA template,thereby producing a cDNA. Thus, an extended probe made in a method setforth herein can be a reverse transcribed DNA molecule. Exemplarymethods for extending nucleic acids are set forth in US Pat. App. Publ.No. US 2005/0037393 A1 or U.S. Pat. No. 8,288,103 or 8,486,625, each ofwhich is incorporated herein by reference.

All or part of a target nucleic acid that is hybridized to a nucleicacid probe can be copied by extension. For example, an extended probecan include at least, 1, 2, 5,10, 25, 50, 100, 200, 500, 1000 or morenucleotides that are copied from a target nucleic acid. The length ofthe extension product can be controlled, for example, using reversiblyterminated nucleotides in the extension reaction and running a limitednumber of extension cycles. The cycles can be run as exemplified for SBStechniques and the use of labeled nucleotides is not necessary.Accordingly, an extended probe produced in a method set forth herein caninclude no more than 1000, 500, 200, 100, 50, 25, 10, 5, 2 or 1nucleotides that are copied from a target nucleic acid. Of courseextended probes can be any length within or outside of the ranges setforth above.

Although the methods of the present disclosure are exemplified by anembodiment where probes that are hybridized to target nucleic acids areextended to copy at least a portion of the target nucleic acid, it willbe understood that the probes can be modified in alternative ways. Theprobes that are hybridized to target nucleic acids can be subjected to areaction that creates a target specific modification of the probe. Atarget specific modification will result only when the probe interactswith a target nucleic acid, for example, via complementary basedhybridization. In many embodiments, the target specific modificationwill be specific to the sequence of the particular target nucleic acidthat interacts with the probe. Examples of useful target specificmodifications, include but are not limited to, insertion or addition ofa sequence by ligation or transposition (see, for example, US Pat. App.Publ. No. 2010/0120098 A1, incorporated herein by reference), chemicalmodifications such as psoralen crosslinking or addition of a detectabletag moiety, modifications by nucleic acid enzymes, ligation of a hairpinlinker, or other modifications set forth in the nucleic acid assays ofUS Pat. App. Publ. No. US 2005/0037393 A1 or U.S. Pat. Nos. 8,288,103 or8,486,625, each of which is incorporated herein by reference.

It will be understood that probes used in a method, composition orapparatus set forth herein need not be nucleic acids. Other moleculescan be used such as proteins, carbohydrates, small molecules, particlesor the like. Probes can be a combination of a nucleic acid component(e.g. having a barcode, primer binding site, cleavage site and/or othersequence element set forth herein) and another moiety (e.g. a moietythat captures or modifies a target nucleic acid).

A method set forth herein can further include a step of acquiring animage of a biological specimen that is in contact with a solid support.The solid support can be in any of a variety of states set forth herein.For example, the solid support can include attached nucleic acid probesor clusters derived from attached nucleic acid probes. Alternatively,the solid support may not include nucleic acid probes, instead being ina state that precedes attachment of nucleic acid probes or in a statethat follows removal of nucleic acid probes from the solid support.Accordingly, an image can be obtained at any of a variety of points in amethod set forth herein.

An image can be obtained using detection devices known in the art.Examples include microscopes configured for light, bright field, darkfield, phase contrast, fluorescence, reflection, interference, orconfocal imaging. A biological specimen can be stained prior to imagingto provide contrast between different regions or cells. In someembodiments, more than one stain can be used to image different aspectsof the specimen (e.g. different regions of a tissue, different cells,specific subcellular components or the like). In other embodiments, abiological specimen can be imaged without staining.

In particular embodiments, a fluorescence microscope (e.g. a confocalfluorescent microscope) can be used to detect a biological specimen thatis fluorescent, for example, by virtue of a fluorescent label.Fluorescent specimens can also be imaged using a nucleic acid sequencingdevice having optics for fluorescent detection such as a GenomeAnalyzer®, MiSeq®, NextSeq® or HiSeq® platform device commercialized byIllumina, Inc. (San Diego, Calif.); or a SOLiD™ sequencing platformcommercialized by Life Technologies (Carlsbad, Calif.). Other imagingoptics that can be used include those that are found in the detectiondevices described in Bentley et al., Nature 456:53-59 (2008), PCT Publ.Nos. WO 91/06678, WO 04/018497 or WO 07/123744; U.S. Pat. Nos.7,057,026, 7,329,492, 7,211,414, 7,315,019 or 7,405,281, and US Pat.App. Publ. No. 2008/0108082, each of which is incorporated herein byreference.

An image of a biological specimen can be obtained at a desiredresolution, for example, to distinguish tissues, cells or subcellularcomponents. Accordingly, the resolution can be sufficient to distinguishcomponents of a biological specimen that are separated by at least 0.5μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, 1 mm or more.Alternatively or additionally, the resolution can be set to distinguishcomponents of a biological specimen that are separated by at least 1 mm,500 μm, 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm or less.

A method set forth herein can include a step of correlating locations inan image of a biological specimen with barcode sequences of nucleic acidprobes that are attached to a surface to which the biological specimenis, was or will be contacted. Accordingly, characteristics of thebiological specimen that are identifiable in the image can be correlatedwith the nucleic acids that are found to be present in their proximity.Any of a variety of morphological characteristics can be used in such acorrelation, including for example, cell shape, cell size, tissue shape,staining patterns, presence of particular proteins (e.g. as detected byimmunohistochemical stains) or other characteristics that are routinelyevaluated in pathology or research applications. Accordingly, thebiological state of a tissue or its components as determined by visualobservation can be correlated with molecular biological characteristicsas determined by spatially resolved nucleic acid analysis.

A solid support upon which a biological specimen is imaged can includefiducial markers to facilitate determination of the orientation of thespecimen or the image thereof in relation to probes that are attached tothe solid support. Exemplary fiducials include, but are not limited tobeads (with or without fluorescent moieties or moieties such as nucleicacids to which labeled probes can be bound), fluorescent moleculesattached at known or determinable features, or structures that combinemorphological shapes with fluorescent moieties. Exemplary fiducials areset forth in US Pat. App. Publ. No. 2002/0150909 A1or U.S. patentapplication Ser. No. 14/530,299, each of which is incorporated herein byreference. One or more fiducials are preferably visible while obtainingan image of a biological specimen. Preferably, the solid supportincludes at least 2, 3, 4, 5, 10, 25, 50, 100 or more fiducial markers.The fiducials can be provided in a pattern, for example, along an outeredge of a solid support or perimeter of a location where a biologicalspecimen resides. In a preferred embodiment, one or more fiducials aredetected using the same imaging conditions used to visualize abiological specimen. However if desired separate images can be obtained(e.g. one image of the biological specimen and another image of thefiducials) and the images can be aligned to each other.

Optionally, a biological specimen, can be removed from a solid supportafter an image has been obtained and after target nucleic acids havebeen captured by nucleic acid probes on the solid support. Thus, amethod of the present disclosure can include a step of washing a solidsupport to remove cells, tissue or other materials from a biologicalspecimen. Removal of the specimen can be performed using any suitabletechnique and will be dependent on the tissue sample. In some cases, thesolid support can be washed with water. The water can contain variousadditives, such as surfactants (e.g. detergents), enzymes (e.g.proteases and collagenases), cleavage reagents, or the like, tofacilitate removal of the specimen. In some embodiments, the solidsupport is treated with a solution comprising a proteinase enzyme.Alternatively or additionally, the solution can include cellulase,hemicelluase or chitinase enzymes (e.g. if desiring to remove a tissuesample from a plant or fungal source). In some cases, the temperature ofa wash solution will be at least 30° C., 35° C., 50° C., 60° C. or 90°C. Conditions can be selected for removal of a biological specimen whilenot denaturing hybrid complexes formed between target nucleic acids andsolid support-attached nucleic acid probes.

A method of the present disclosure can further include a step ofremoving one or more extended probes from a solid support. In particularembodiments, the probes will have included a cleavage site such that theproduct of extending the probes will also include the cleavage site.Alternatively, a cleavage site can be introduced into a probe during amodification step. For example a cleavage site can be introduced into anextended probe during the extension step.

Exemplary cleavage sites include, but are not limited to, moieties thatare susceptible to a chemical, enzymatic or physical process thatresults in bond breakage. For example, the location can be a nucleotidesequence that is recognized by an endonuclease. Suitable endonucleasesand their recognition sequences are well known in the art and in manycases are even commercially available (e.g. from New England Biolabs,Beverley Mass.; ThermoFisher, Waltham, Mass. or Sigma Aldrich, St. LouisMo.). A particularly useful endonuclease will break a bond in a nucleicacid strand at a site that is 3′-remote to its binding site in thenucleic acid, examples of which include Type II or Type Ils restrictionendonucleases. In some embodiments an endonuclease will cut only onestrand in a duplex nucleic acid (e.g. a nicking enzyme). Examples ofendonucleases that cleave only one strand include Nt.BstNBI and Nt.Alwl.

In some embodiments, a cleavage site is an abasic site or a nucleotidethat has a base that is susceptible to being removed to create an abasicsite. Examples of nucleotides that are susceptible to being removed toform an abasic site include uracil and 8-oxo-guanine. Abasic sites canbe created by hydrolysis of nucleotide residues using chemical orenzymatic reagents. Once formed, abasic sites may be cleaved (e.g. bytreatment with an endonuclease or other single-stranded cleaving enzyme,exposure to heat or alkali), providing a means for site-specificcleavage of a nucleic acid. An abasic site may be created at a uracilnucleotide on one strand of a nucleic acid. The enzyme uracil DNAglycosylase (UDG) may be used to remove the uracil base, generating anabasic site on the strand. The nucleic acid strand that has the abasicsite may then be cleaved at the abasic site by treatment withendonuclease (e.g. EndoIV endonuclease, AP lyase, FPG glycosylase/APlyase, EndoVIII glycosylase/AP lyase), heat or alkali. In a particularembodiment, the USER™ reagent available from New England Biolabs is usedfor the creation of a single nucleotide gap at a uracil base in anucleic acid.

Abasic sites may also be generated at non-natural/modifieddeoxyribonucleotides other than uracil and cleaved in an analogousmanner by treatment with endonuclease, heat or alkali. For example,8-oxo-guanine can be converted to an abasic site by exposure to FPGglycosylase. Deoxyinosine can be converted to an abasic site by exposureto AlkA glycosylase. The abasic sites thus generated may then becleaved, typically by treatment with a suitable endonuclease (e.g.EndoIV or AP lyase).

Other examples of cleavage sites and methods that can be used to cleavenucleic acids are set forth, for example, in U.S. Pat. No. 7,960,120,which is incorporated herein by reference.

Modified nucleic acid probes (e.g. extended nucleic acid probes) thatare released from a solid support can be pooled to form a fluidicmixture. The mixture can include, for example, at least 10, 100, 1×10³,1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹ or more different modifiedprobes. Alternatively or additionally, a fluidic mixture can include atmost 1×10⁹, 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 100, 10 or fewerdifferent modified probes. The fluidic mixture can be manipulated toallow detection of the modified nucleic acid probes. For example, themodified nucleic acid probes can be separated spatially on a secondsolid support (i.e. different from the solid support from which thenucleic acid probes were released after having been contacted with abiological specimen and modified), or the probes can be separatedtemporally in a fluid stream.

Modified nucleic acid probes (e.g. extended nucleic acid probes) can beseparated on a solid support in a capture or detection method commonlyemployed for microarray-based techniques or nucleic acid sequencingtechniques such as those set forth previously herein. For example,modified probes can be attached to a microarray by hybridization tocomplementary nucleic acids. The modified probes can be attached tobeads or to a flow cell surface and optionally amplified as is carriedout in many nucleic acid sequencing platforms. Modified probes can beseparated in a fluid stream using a microfluidic device, dropletmanipulation device, or flow cytometer. Typically, detection is carriedout on these separation devices, but detection is not necessary in allembodiments.

A particularly useful droplet manipulation device is a droplet actuatoras described for example in U.S. Pat. Nos. 8,637,242, 6,911,132,entitled “Apparatus for Manipulating Droplets by Electrowetting-BasedTechniques,” issued on Jun. 28, 2005; Pamula et al., U.S. Patent Pub.No. 20060194331, entitled “Apparatuses and Methods for ManipulatingDroplets on a Printed Circuit Board,” published on Aug. 31, 2006;Pollack et al., International Patent Pub. No. WO/2007/120241, entitled“Droplet-Based Biochemistry,” published on Oct. 25, 2007; Shenderov,U.S. Pat. No. 6,773,566, entitled “Electrostatic Actuators forMicrofluidics and Methods for Using Same,” issued on Aug. 10, 2004;Shenderov, U.S. Pat. No. 25 6,565,727, entitled “Actuators forMicrofluidics Without Moving Parts,” issued on May 20, 2003; Kim et al.,U.S. Patent Pub. No. 20030205632, entitled “ElectrowettingdrivenMicropumping,” published on Nov. 6, 2003; Kim et al., U.S. Patent Pub.No. 20060164490, entitled “Method and Apparatus for Promoting theComplete Transfer of Liquid Drops from a Nozzle,” published on Jul. 27,2006; Kim et al., U.S. Patent Pub. No. 20070023292, entitled “SmallObject Moving on Printed Circuit Board,” published on Feb. 1, 2007; Shahet al., U.S. Patent Pub. No. 20090283407, entitled “Method for UsingMagnetic Particles in Droplet Microfluidics,” published on Nov. 19,2009; Kim et al., U.S. Patent Pub. No. 20100096266, entitled “Method andApparatus for Real-time Feedback Control of Electrical Manipulation ofDroplets on Chip,” published on Apr. 22, 2010; Velev, U.S. Pat. No.7,547,380, entitled “Droplet Transportation Devices and Methods Having aFluid Surface,” issued on Jun. 16, 2009; Sterling et al., U.S. Pat. No.7,163,612, entitled “Method, Apparatus and Article for MicrofluidicControl via Electrowetting, for Chemical, Biochemical and BiologicalAssays and the Like,” issued on Jan. 16, 2007; Becker et al., U.S. Pat.No. 7,641,779, entitled “Method and Apparatus for Programmable FluidicProcessing,” issued on Jan. 5, 2010; Becker et al., U.S. Pat. No.6,977,033, entitled “Method and Apparatus for Programmable FluidicProcessing,” issued on Dec. 20, 2005; Decre et al., U.S. Pat. No.7,328,979, entitled “System for Manipulation of a Body of Fluid,” issuedon Feb. 12, 2008; Yamakawa et al., U.S. Patent Pub. No. 15 20060039823,entitled “Chemical Analysis Apparatus,” published on Feb. 23, 2006; Wu,U.S. Patent Pub. No. 20110048951, entitled “Digital Microfluidics BasedApparatus for Heat-exchanging Chemical Processes,” published on Mar. 3,2011; Fouillet et al., U.S. Patent Pub. No. 20090192044, entitled“Electrode Addressing Method,” published on Jul. 30, 2009; Fouillet etal., U.S. Pat. No. 7,052,244, entitled “Device for Displacement of SmallLiquid Volumes Along a Micro-catenary Line by Electrostatic Forces,”issued on May 30, 2006; Marchand et al., U.S. Patent Pub. No.20080124252, entitled “Droplet Microreactor,” published on May 29, 2008;Adachi et al., U.S. Patent Pub. No. 20090321262, entitled “LiquidTransfer Device,” published on Dec. 31, 2009; Roux et al., U.S. PatentPub. No. 20050179746, entitled “Device for Controlling the Displacementof a Drop Between Two or Several Solid Substrates,” published on Aug.18, 2005; and Dhindsa et al., “Virtual Electrowetting Channels:Electronic Liquid Transport with Continuous Channel Functionality,” LabChip,10:832-836 (2010), each of which is incorporated herein byreference.

Modified probes (e.g. extended nucleic acid probes) can be detected, forexample, following separation from a fluidic mixture using methods setforth above or known in the art. In particular embodiments, modifiedprobes that are separated on a second solid support (i.e. a solidsupport that is different from the first solid support where contact wasmade between probes and biological specimen) can be detected usingmicroarray-based techniques or nucleic acid sequencing techniques suchas those set forth previously herein. Probes that are separated in afluid stream can be detected using optical, electrical or otherdetectors that are outfitted in known microfluidic devices, dropletmanipulation devices, or flow cytometers. A detection method can be usedto determine target nucleic acid sequences, barcode sequences or othersequence regions of extended probes.

Several embodiments have been exemplified with regard to removingmodified probes from the solid support where the probes were produced.However, it will be understood that probes on a solid support can becontacted with a biological specimen, modified on the solid support inthe presence of target nucleic acids from the specimen and then themodified probes can be detected on the solid support. In such anembodiment, the biological specimen can be removed from the solidsupport prior to the detection step.

In particular embodiments the present disclosure provides a method forspatially tagging nucleic acids of a biological specimen that includesthe steps of (a) providing a plurality of nucleic acid primers attachedto a solid support, wherein the nucleic acid primers in the pluralityinclude a universal primer sequence that is common to the nucleic acidprimers in the plurality; (b) binding a population of nucleic acidprobes to the plurality of nucleic acid primers, wherein the nucleicacid probes include a universal primer binding sequence that hybridizesto the universal primer sequence, a target capture sequence and abarcode sequence that differs from barcode sequences of other nucleicacid probes in the population, thereby attaching the different nucleicacid probes at randomly located positions on the solid support; (c)amplifying the different nucleic acid probes by extension of the nucleicacid primers, thereby producing nucleic acid clusters having copies ofthe barcode sequence and target capture sequence at the randomly locatedpositions on the solid support; (d) performing a sequencing reaction todetermine the barcode sequences at the randomly located positions on thesolid support; (e) contacting a biological specimen with the nucleicacid clusters on the solid support; (f) hybridizing the target capturesequences of the clusters to target nucleic acids from portions of thebiological specimen that are proximal to the clusters; and (g) extendingthe target capture sequences to produce extended probes that includesequences from the target nucleic acids and the copies of the barcodesequences, thereby tagging the nucleic acids of the biological specimen.

As exemplified previously herein, a plurality of nucleic acid primerscan be attached to a solid support, wherein the nucleic acid primers inthe plurality include a universal primer sequence that is common to thenucleic acid primers in the plurality. In this embodiment, a secondplurality of nucleic acid primers can be attached to the solid support,and the nucleic acid primers in the second plurality can have a seconduniversal primer sequence that is common to the nucleic acid primers inthe second plurality. In this embodiment, a plurality of differentnucleic acid probes that is contacted with the support can include auniversal primer binding sequence that hybridizes to the universalprimer on the solid support, as set forth above, and the differentnucleic acid probes can also include a second universal primer bindingsequence that hybridizes to the second universal primer sequence. Thisconfiguration of universal primers and universal primer binding sitescan be particularly useful for amplifying the different nucleic acidprobes via bridge amplification, wherein the nucleic acid primers in thefirst and second plurality are extended.

Typically, when a nucleic acid probe contains first and second universalprimer binding sites, they will be located at the ends of the probe. Insome embodiments it may be desirable to remove at least one of theprimer binding sites from the nucleic acid probe or from ampliconsproduced from the probe. Accordingly, the nucleic acid probes canoptionally include a cleavage site between the target capture sequenceand one of the universal primer binding sequence. In this case, acleavage reaction can be performed to separate the universal primerbinding site from the target capture sequence. Generally, the portion ofthe probe (or its amplicons) that contains the target capture sequencewill be attached to the solid support resulting in removal of the primerbinding site from the solid support and retention of the target capturesequence. Thus, the cleaved probe can be used for hybridizing targetnucleic acids and the cleaved probe can be extended using method setforth previously herein.

In some embodiments, a nucleic acid probe will include two differentcleavage sites. A first cleavage site will be located between a firstprimer binding site and one or more other sequence elements of theprobe. A second cleavage site can be located between a second primerbinding site and the one or more other sequence elements of the probe.The cleavage sites can be reactive to different cleavage reactions suchthat each one can be selectively cleaved without necessarily cleavingthe other. Accordingly, the first cleavage site can be cleaved prior tomodifying the probe (for example, prior to producing an extended probe),thereby separating the first primer binding site from the one or moreother sequence elements that remain attached to a solid support. Thesecond cleavage site can be cleaved after modifying the probe (forexample, after producing the extended probe), thereby releasing themodified probe for subsequent detection.

Alternatively, a nucleic acid probe can include the first cleavage siteand a primer that is used to capture or amplify the nucleic acid probecan include the second cleavage site. In this configuration, the firstcleavage site can be located between a first primer binding site and oneor more other sequence elements of the probe such that cleavageseparates the first primer binding site from one or more other sequenceelements of the probe that remain attached to a solid support. Again,this first cleavage step will typically be carried out prior tomodifying the probe (for example, prior to producing an extended probe).A second cleavage step can be carried out to cleave the second cleavagesite after modifying the probe (for example, after producing theextended probe), thereby releasing the modified probe for subsequentdetection.

The two embodiments above exemplify a cleavage site located between apoint of attachment of a nucleic acid probe (or modified nucleic acidprobe) and one or more sequences of the probe (or modified probe) thatcontain information such as a spatial barcode or target sequence. Thus,this cleavage site is useful for release of modified probes (e.g.extended probes) to detect the sequence information and determine whatsequences are present in a biological specimen and where the sequencesare present in the specimen.

In some embodiments, one or more probes that are contacted with a solidsupport in a method set forth herein can include a sequencing primerbinding site. Accordingly, a modified probe (e.g. extended probe) can bedetected in a sequencing technique that includes a step of hybridizing asequencing primer to the sequencing primer binding site. The sequencingprimer binding site can be located in the probe such that cleavage of amodified version of the probe (e.g. an extended probe) will yield areleased probe that includes the sequencing primer binding site. Thesequencing primer binding site can be a universal sequencing primerbinding site such that a plurality of different probes (e.g. havingdifferent barcode and/or target sequences) will have the same sequencingprimer binding site.

This disclosure further provides a method for spatially tagging nucleicacids of a biological specimen, the method including steps of (a)providing an array of beads on a solid support, wherein differentnucleic acid probes are attached to different beads in the array,wherein the different nucleic acid probes each include a barcodesequence, wherein each bead includes a different barcode sequence fromother beads on the solid support, and wherein each of the differentnucleic acid probes includes a target capture sequence; (b) performing adecoder probe hybridization reaction on the solid support to determinethe barcode sequences at the randomly located probes on the solidsupport; (c) contacting a biological specimen with the array of beads;(d) hybridizing the different nucleic acid probes to target nucleicacids from portions of the biological specimen that are proximal to thebeads; and (e) extending the different nucleic acid probes to produceextended probes that include sequences from the target nucleic acids andthe barcode sequences, thereby tagging the nucleic acids of thebiological specimen.

It will be understood that manipulations of solid supports or of nucleicacids attached to solid supports can be carried out using beads as solidsupports. The beads can be attached to a surface (e.g. an array of wellsas in a BeadArrayTM from Illumina) before or after such manipulationsare carried out. For example, nucleic acid probes can be captured onbeads before or after the beads are distributed on an array, nucleicacid probes can be amplified to create amplicons on beads before orafter the beads are distributed on an array etc.

EXAMPLE I Spatially Tagging mRNA from a Tissue Sample Using IlluminaFlow Cells

A method for generating barcoded oligo-dT containing clusters, thenrevealing the barcoded oligo-dT with a restriction enzyme digestfollowed by sequencing is described in FIG. 1. A library of fragmentscontaining a single stranded, barcoded oligo-dA, P5′,P7, SBS3 sequencingprimer binding site and a BspH1 restriction enzyme site (shown in thetop panel of FIG. 1) were prepared by oligo synthesis (Integrated DNATechnologies). The barcodes were 27mers and were randomly generatedduring synthesis. The binding site for the SBS3 sequencing primer wasincluded for decoding of the barcode by sequencing. An oligo-dA stretchwas included to generate an oligo dT site upon clustering andlinearization. Bridge amplification and clustering were performedaccording to standard cluster chemistry (Illumina TruSeq PE Cluster Kitv3 cBot P/N: 15037931) on an Illumina GA flow cell using manufacture'srecommended protocol.

Following bridge amplification and clustering the clusters werelinearized by cleavage of 8-oxo-G in P7 primer using FormamidopyrimidineDNA glycosylase (Fpg) enzyme provided in the TruSeq PE Cluster kit. Thiswas followed by restriction enzyme digest with 200 Units/mL BspH1 (NEBCat # R0517L at 37° C. for 15 min to remove P7′ from the P5 adapteranchored strand of the cluster to unveil the oligo-dT stretch forsubsequent extension in the presence of an mRNA. Enzyme concentrationsin the range of 100-400 U/mL have been tested for 15 or 30 min. Thede-coding of the barcode was initiated by the SBS3 sequencing primer.

As shown in the bottom panel of FIG. 1, oligo-dT sequences in thecluster were used to capture poly A+RNA after decoding of the barcode.Barcoded cDNA was produced by extension of the oligo-dT strand of thecluster using TruSeq RNA Sample Prep Kit (Illumina P/N: 15012997) andMMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Epicentre P/N:MM070150) according to the manufacturer's recommended conditions. Thecaptured RNA was used as a template. Barcoded cDNA was released from theP5 sequence of the flow cell using Illumina's Uracil Specific Excisionreagents (USER) (Illumina's TruSeq PE cluster kit) liberating a barcodedcDNA library that was used for sequencing on a second Illumina flowcell.

The availability of oligo dT capture sequence after the restrictionenzyme digest with BspH1 was confirmed by hybridizing the linearizedclusters with a Cy5 labeled poly A (24mer) as diagrammed in panel A ofFIG. 2. Briefly, after the restriction enzyme digestion, the clusterswere treated with 0.1N NaOH and washed with HT2 low salt buffer toremove the second strand on the flow cell. Then, 500 nM of Cy5 oligo-dA(24mer) was flowed over the linearized and denatured clusters at 30μl/min rate and incubated at 40° C. for 5 min and then imaged.Hybridization of Cy5 labeled poly A to the oligo dT was detected inlanes 2-7 of the GA flow cell where the oligo dT containing BODT-1libraries were present (see the image of the flow cell shown in FIG. 2,Panel B). As evident from the flow cell image (Panel B), and thebargraph (Panel C), the control PhiX libraries (lanes 1 and 8of the flowcell) were shown to have very low fluorescence in the Cy5 signal. Theseresults demonstrated that an oligo-dT site can be created in the clusterthat upon linearization can bind specifically to Cy5 poly A (24mer).

The sequencing metrics of the flow cell described above with 3.2 μM ofBODT-1 library is given in the table shown in FIG. 3. Millions of readswere detected in 21 tiles from GA sequencing. Following sequencing, thenumber of unique barcodes were determined as plotted in FIG. 4. This wasdone by assuming that every passing filter (PF) read was a barcode anddetermining the number of unique reads (barcodes) in each lane. Between5 and 11 million unique barcoded clusters were detected after sequencingtiles compared to the PhiX control libraries. These results demonstratedthat sequence decoding of a library of barcoded oligo-dT sequences isfeasible and generates millions of unique barcodes.

EXAMPLE II Cell Adhesion on Illumina Flow Cells

Single cells were captured on a patterned flow cell (HiSeq X10 flowcell, Illumina). All reagent flow steps were performed using aperistaltic pump or the cBOT cluster generation instrument (Illumina).Briefly, nuclease free water was flowed on all lanes of the patternedflow cell followed by 30-70K Poly D Lysine Solution (100 μg/ml and 20μg/ml) at a flow rate of 100 μl/min for 8 min. Heat inactivated FetalBovine Serum (Life Technologies #10082-139) was also tested as anadhesive. The adhesives were incubated on the flow cell lanes for 1 hr,followed by a 1× PBS+0.5% Pluronic F-68 (Life Technologies #24040-032)wash. Next, the cells were adhered to the coated flow cells by flowing 5to 50 cells/μl or approximately 100-1000 cells per lane at a rate of 100μl/min, followed by an incubation step for 60 min to bind the cells. Theflow cell was washed with 1× PBS/0.5% pluronic at a rate of 75 μl/min.If cells were fixed on the flow cell, 1% Paraformaldehyde (PFA) wasflowed on the flow cell after flowing the cells as described above andincubated for 15 min followed by the 1× PBS/0.5% pluronic was step. Theflow cell was removed and the number of cells per lane counted using amicroscope.

FIG. 5, Panel A shows an image of cells captured on the patterned flowcell. The cell count data shown in FIG. 5, Panel B confirmed that thepoly D Lysine coated flow cells aided cell adherence compared to the BSAcoated or no adhesive treated control. As shown in FIG. 6, the adheredcells can be successfully fixed with 1% PFA.

EXAMPLE Ill Spatially Localized Capture of Target mRNA by ProbesAttached to a Gel Surface

This example describes creation of a lawn of poly T probes on a gelcoated slide, placement of tissue slices on top of a lawn of poly Tprobes, release of RNA from the tissue sections, capture of the releasedmRNA by the poly T probes, reverse transcription to Cy 3 label the polyT probes, removal of the tissue and imaging of the slide.

FIG. 7, Panel A shows a diagrammatic representation of steps andreagents used to create probes attached to a gel. Briefly, a microscopeslide was coated with silane free acrylamide (SFA), P5 and P7 primerswere attached (see US Pat. App. Pub. No. 2011/0059865 A1, which isincorporated herein by reference), probes having a poly A sequence andeither a P5 or P7 complementary sequence were hybridized to the P5 andP7 primers, respectively, and the P5 and P7 primers were extended toproduce poly T sequence extensions. A quality control step was performedby hybridizing Cy5 labeled polyA oligonucleotides to the extendedprimers and imaging the surface using an Axon Imager.

As shown in Panel B of FIG. 7, a tissue section was placed on the gelhaving the polyT extended primers. The tissue was treated to releasemRNA and poly A tails of the released mRNA were hybridized to poly Tsequences of the extended primers. The poly T sequences were extendedusing the captured mRNAs as templates and the extended primers wereselectively labeled with Cy3. The tissue was removed from the gel andthe surface was imaged to detect Cy3 flourescence.

As shown in the image of FIG. 7, areas of the gel that were proximal toareas of the tissue that released mRNA species appeared fluorescentwhile areas that did not release mRNA appeared dark in the image. Thus,the captured mRNA created a fingerprint-like image of the tissue.

EXAMPLE IV Spatially Localized Capture of Target mRNA by Probes Attachedto a BeadArray™ Surface

This example describes placement of tissue slices on top of a BeadArray™having poly T probes, release of RNA from the tissue sections, captureof the released mRNA by the poly T probes, reverse transcription to Cy5label the poly T probes, removal of the tissue and imaging of theBeadArray™.

As shown in Panel A of FIG. 8, a mouse olfactory tissue section wasplaced on a BeadArray™ having polyT probes. The tissue was treated torelease mRNA and poly A tails of the released mRNA were hybridized topoly T sequences of the probes. The poly T sequences were extended usingthe captured mRNAs as templates and the extended primers wereselectively labeled with Cy5. The tissue was removed from the BeadArray™and the BeadArray™ was imaged to detect Cy5 flourescence.

As shown in Panel B of FIG. 7, areas of the BeadArray™ that wereproximal to areas of the tissue that released mRNA species appearedfluorescent while areas that did not release mRNA appeared dark in theimage. Thus, the captured mRNA created a fingerprint-like image of thetissue.

Throughout this application various publications, patents or patentapplications have been referenced. The disclosure of these publicationsin their entireties are hereby incorporated by reference in thisapplication.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

1.-105. (canceled)
 106. A method for spatially tagging nucleic acids ofa biological specimen, comprising: (a) attaching different nucleic acidprobes to a solid support to produce randomly located probes on thesolid support, wherein the different nucleic acid probes each comprise abarcode sequence, and wherein each of the randomly located probescomprise different barcode sequences from other randomly located probeson the solid support; (b) performing a nucleic acid detection reactionon the solid support to determine the barcode sequences of the randomlylocated probes on the solid support; (c) contacting a biologicalspecimen with the solid support that comprises the randomly locatedprobes; (d) hybridizing the randomly located probes to target nucleicacids from portions of the biological specimen that are proximal to therandomly located probes; and (e) extending the randomly located probesto produce extended probes that comprise the barcode sequences andsequences from the target nucleic acids, thereby spatially tagging thenucleic acids of the biological specimen.
 107. The method of claim 106,wherein step (a) further comprises amplifying each of the randomlylocated probes on the solid support to form a cluster that comprisesmultiple amplicons of the nucleic acid probes on the solid support. 108.The method of claim 107, wherein the solid support comprises a pluralityof the clusters and the average pitch for the plurality of clusters isless than 10 microns and/or the average area of the clusters is lessthan 100 um'.
 109. The method of claim 106, wherein the nucleic aciddetection reaction comprises a sequencing reaction or a decoder probehybridization reaction.
 110. The method of claim 106, wherein the solidsupport comprises an array of beads, wherein the different nucleic acidprobes are attached to different beads in the array, wherein step (a)comprises randomly distributing the different beads on the solid supportor wherein the solid support comprises wells having dimensions thataccommodate no more than a single bead of said plurality of beads,wherein the different beads are attached to different barcode sequencesand wherein the number of different barcode sequences exceeds the numberof wells, further wherein the solid support comprises at least 1×10⁶ ofthe different beads that comprise the different nucleic acid probesand/or wherein the array of beads comprises an average pitch of lessthan 10 microns and/or wherein the beads comprise an average diameter ofless than 10 microns.
 111. The method of claim 106, wherein theattaching of the different nucleic acid probes to the solid supportcomprises contacting a fluidic mixture of the different nucleic acidprobes with the solid support, wherein the solid support comprises apattern of discrete features and wherein the different nucleic acidprobes are randomly distributed to discrete features in the pattern, andwherein the number of different barcode sequences exceeds the number offeatures that are contacted by the fluidic mixture and/or wherein thesolid support comprises at least 1×10⁶ of the different nucleic acidprobes.
 112. The method of claim 106, wherein the solid supportcomprises a gel coating, wherein a plurality of nucleic acid primers isattached to the gel coating, wherein the nucleic acid primers in theplurality comprise a universal primer sequence that is common to thenucleic acid primers in the plurality, wherein the different nucleicacid probes comprise a universal primer binding sequence that hybridizesto the universal primer sequence.
 113. The method of claim 106, whereinstep (a) further comprises amplifying the different nucleic acid probesby extension of the nucleic acid primers to produce nucleic acidclusters that comprise multiple amplicons of the nucleic acid probes onthe solid support, and wherein a second plurality of nucleic acidprimers is attached to the gel coating, and wherein the nucleic acidprimers in the second plurality comprise a second universal primersequence that is common to the nucleic acid primers in the secondplurality, wherein the different nucleic acid probes comprise a seconduniversal primer binding sequence that hybridizes to the seconduniversal primer sequence and wherein the amplifying comprises bridgeamplification.
 114. The method of claim 106, wherein the differentnucleic acid probes comprise different target capture sequences thathybridize to different target nucleic acid sequences from the biologicalspecimen.
 115. The method of claim 106, wherein the different nucleicacid probes comprise a target capture sequence that is common to therandomly located probes on the solid support, and wherein the targetcapture sequence comprises a poly T or poly A sequence.
 116. The methodof claim 106, further comprising a step of acquiring an image of thebiological specimen in contact with the solid support, and a step ofcorrelating the barcode sequences determined at the randomly locatedprobes with locations in the image of the biological specimen.
 117. Themethod of claim 116, further comprising removing the extended probesfrom the solid support, and pooling the extended probes to form amixture of the extended probes that have been removed from the solidsupport and/or determining the sequences from the target nucleic acidsand the barcode sequences for the extended probes that have been removedfrom the solid support,
 118. The method of claim 117, further comprisingattaching the extended probes that have been removed from the solidsupport to a second solid support,
 119. The method of claim 117, whereinthe sequences from the target nucleic acids and the barcode sequencesare determined by a sequencing-by-synthesis technique.
 120. The methodof claim 106, wherein the solid support is located in or on a flow cellduring step (b), and wherein the solid support is removed from the flowcell during step (c) or the flow cell is opened to expose the solidsupport during step (c).
 121. The method of claim 106, wherein thebiological specimen that is contacted with the solid support is amixture of cells, and wherein step (c) further comprises attaching thecells to the solid support and/or lysing the cells to release the targetnucleic acids from the cells.
 122. The method of claim 106, wherein thebiological specimen that is contacted with the solid support is atissue, and wherein step (c) further comprises attaching the tissue tothe solid support and/or permeabilizing the tissue to release the targetnucleic acids from the tissue.
 123. The method of claim 106, wherein thetarget nucleic acid is selected from the group consisting of mRNA, gDNA,rRNA or tRNA.
 124. A method for spatially tagging nucleic acids of abiological specimen, comprising: (a) providing a plurality of nucleicacid primers attached to a solid support, wherein the nucleic acidprimers in the plurality comprise a universal primer sequence that iscommon to the nucleic acid primers in the plurality; (b) binding apopulation of nucleic acid probes to the plurality of nucleic acidprimers, wherein the nucleic acid probes comprise a universal primerbinding sequence that hybridizes to the universal primer sequence, atarget capture sequence and a barcode sequence that differs from barcodesequences of other nucleic acid probes in the population, therebyattaching the different nucleic acid probes at randomly locatedpositions on the solid support; (c) amplifying the different nucleicacid probes by extension of the nucleic acid primers, thereby producingnucleic acid clusters having copies of the barcode sequence and targetcapture sequence at the randomly located positions on the solid support;(d) performing a sequencing reaction to determine the barcode sequencesat the randomly located positions on the solid support; (e) contacting abiological specimen with the nucleic acid clusters on the solid support;(f) hybridizing the target capture sequences of the clusters to targetnucleic acids from portions of the biological specimen that are proximalto the clusters; and (g) extending the target capture sequences toproduce extended probes that comprise sequences from the target nucleicacids and the copies of the barcode sequences, thereby tagging thenucleic acids of the biological specimen.
 125. The method of claim 124,wherein a second plurality of nucleic acid primers is attached to thesolid support, wherein the nucleic acid primers in the second pluralitycomprise a second universal primer sequence that is common to thenucleic acid primers in the second plurality.
 126. The method of claim125, wherein the different nucleic acid probes comprise a seconduniversal primer binding sequence that hybridizes to the seconduniversal primer sequence.
 127. The method of claim 126, wherein theamplifying of the different nucleic acid probes comprises bridgeamplification wherein nucleic acid primers in the first and secondplurality are extended.
 128. A method for spatially tagging nucleicacids of a biological specimen, comprising: (a) providing an array ofbeads on a solid support, wherein different nucleic acid probes areattached to different beads in the array, wherein the different nucleicacid probes each comprise a barcode sequence, wherein each beadcomprises a different barcode sequences from other beads on the solidsupport, and wherein each of the different nucleic acid probes comprisesa target capture sequence; (b) performing a decoder probe hybridizationreaction on the solid support to determine the barcode sequences at therandomly located probes on the solid support; (c) contacting abiological specimen with the array of beads; (d) hybridizing thedifferent nucleic acid probes to target nucleic acids from portions ofthe biological specimen that are proximal to the beads; and (e)extending the different nucleic acid probes to produce extended probesthat comprise sequences from the target nucleic acids and the barcodesequences, thereby tagging the nucleic acids of the biological specimen.129. The method of claim 128, wherein step (a) comprises randomlydistributing the different beads on the solid support.
 130. The methodof claim 128, wherein the solid support comprises wells havingdimensions that accommodate no more than a single bead of said pluralityof beads and wherein the number of different barcode sequences exceedsthe number of wells.